Hybridization Reaction Research Articles

Probing the Contributions to DNA Hybridization Dynamics with Time-Resolved Infrared Spectroscopy

DNA can undergo a diverse set of folding and hybridization transitions of which the energetics and dynamics are highly sensitive to the local solution environment as well as nucleobase chemical modifications. Such sensitivity is used to perform various biological functions and can fuel many nanotechnology applications. However, the physical properties of folding and hybridization reactions that underly these applications often remain poorly understood. DNA folding and hybridization is complicated by many fast structural motions, such as fraying and sliding, and therefore investigation of DNA folding and hybridization requires the ability to probe DNA structure across both fast (ns-µs) and slow (ms-s) timescales. infrared (IR) spectroscopy offers the capability to probe dynamics from ultrafast timescales to seconds with nucleobase-specific information. Along these lines our group has developed temperature-jump (T-jump) IR and two-dimensional IR (2D IR) methods that can monitor DNA interactions from nanoseconds to many seconds. We are investigating DNA unfolding and duplex dehybridization and how they are modulated by nucleobase sequence, chemical modifications, and nucleobase protonation using T-jump IR spectroscopy. Recently, we have found that the modified nucleobases 5-formyl- and 5-carboxylcytosine can alter DNA base pairing and hybridization with great sensitivity to solution pH. Our results provide insight into a complex interplay between sequence, chemical modification, and sequence that may tune DNA hybridization dynamics.

First evaluation of the automated-multiplex-PCR Unyvero ITI G2 cartridge for rapid diagnosis of osteo-articular infections

ObjectiveConventional microbiological methods (CMM), including long-term culture, for the diagnosis of osteo-articular infections (OAI) fail in at least 5% of all cases. Only one IOA dedicated molecular method has been commercialized, and only the first version of this kit has been studied. The aim of this work was to evaluate the concordance between test results obtained with the second version of the Unyvero ITI G2 cartridge (Curetis) and CMM. The cartridge, combining one-step automated lysis/DNA extraction with multiplex PCR and amplicon detection by array hybridization, allows for the detection of 102 prevalent pathogens and their antibiotic resistance markers directly in clinical specimens (liquid [n=8] or solid [n=32]). Material and methodsFrozen samples from 40 patients who underwent orthopedic surgery at Pitié-Salpêtrière hospital were tested retrospectively with the cartridge: 5 were culture-negative, 25 revealed monomicrobial and 10 polymicrobial OAI. The 2 main surgical sites were hip (22.5%) and knee (17.5%). ResultsExtraction, amplification and hybridization reactions were completed in 28 of the 40 cases, failed in all cartridge chambers in 6 cases, and in 1 or 2 chambers in an additional 6 cases. Overall sensitivity and specificity for microorganism identification were estimated at 67.6% and 98.2%, when complete and partial failures were excluded. ConclusionsThese results show that the performances of the second version of the Unyvero ITI G2 cartridge should be further enhanced before considering avoiding conventional microbiological methods.

Studies on the interactions of Ag(i) with DNA and their implication on the DNA-templated synthesis of silver nanoclusters and on the interaction with complementary DNA and RNA sequences.

Silver nanoclusters (AgNCs) prepared by the reduction of silver ions in the presence of DNA oligonucleotides have attracted great interest as potential diagnostic tools for their tunable and high fluorescent properties. In this work, three DNA sequences that consist of a 12-nucleotide long probe sequence at the 5′-end linked to the complementary sequence to three miRNAs are studied. First, the interaction of these sequences with Ag(i) was characterized by means of circular dichroism spectroscopy. By applying multivariate methods to the analysis of spectroscopic data, two complexes with different Ag(i) : DNA ratios were resolved. Secondly, the impact of several experimental variables, such as temperature, borohydride concentration and reaction time, on the formation of AgNCs templated by these three sequences was studied. Finally, the fluorescence properties of the duplexes formed by DNA probes with complementary DNA or miRNA sequences were studied. The results presented here highlight the role of the secondary structure adopted by the DNA probe on the fluorescence properties of DNA-stabilized AgNCs which, in turn, affect the development of methods for miRNA detection.

A colorimetric biosensor for ultrasensitive detection of the SURF1 gene based on a dual DNA-induced cascade hybridization reaction.

In this work, a simple and ultrasensitive colorimetric biosensor for detection of SURF1 gene fragments (Leigh syndrome) has been developed based on a dual DNA-induced cascade hybridization reaction. Firstly, a biotin labeled capture probe was immobilized on a streptavidin labeled 96-well transparent plate surface. Then the target SURF1 fragment and auxiliary probe S1 were added into the reaction system to form a "Y" structure with the capture probe. Furthermore, to achieve a highly efficient signal amplification strategy, digoxin labeled P1, P2, P3 and P4 probes were used to cause a dual DNA-induced cascade hybridization reaction on the "Y" structure of the 96-well plate surface. As a detection probe, the HRP anti-digoxin antibody was combined on the surface to produce a colorimetric response to the SURF1 fragment in the presence of TMB. Under the optimal conditions, the established method exhibited a wide linear range from 1.0 × 10-13 M to 1.0 × 10-8 M and a detection limit to SURF1 as low as 1.73 × 10-14 M. In addition, the strategy has been successfully applied to the detection of SURF1 in spiked human serum samples. Therefore, the established biosensor has potential application prospects in gene fragment analysis and early diagnosis of clinical diseases.

Sensitive detection of microRNA-21 in cancer cells and human serum with Au@Si nanocomposite and lateral flow assay

We report a highly sensitive approach for detecting microRNA-21 (miR-21) in cancer cells and human serum by using Au@Si nanocomposite labeled lateral flow assay. The Au@Si nanocomposite was prepared by coating numerous 3–5 nm gold nanoparticles (GNP) on a silica nanoparticle (SiNP) with a diameter of 150 nm and used as colored label on the lateral flow assay for signal amplification. TEM results show there are around 1000 GNPs coated on the SiNP surface. The principle of miR-21 detection is based on on-strip DNA-microRNA hybridization reactions to form DNA-miR-21-DNA-Au@Si complexes, which are captured on the test zone of the lateral flow test strip and produce a visible red band. A thiol-modified detecting DNA probe (Det-DNA) and a biotin-modified capturing DNA probe (Cap-DNA), which are complementary to miR-21, were used to prepare the lateral flow test strips. After systematic optimization, the method can detect a minimum concentration of 1.0 pM miR-21, which is 60 times lower than that of the GNP-based lateral flow assay (Gao et al. Biosens & Bioelectro, 2014, 54, 578–584). The method was applied to detect miR-21 in cancer cells and spiked human serum with satisfactory results.

Nanoscale assembly line composed of dual DNA-machines enabling sensitive microRNA detection using upconversion nanoparticles probes

DNA machines are smart artificial devices that perform well-organized DNA hybridization reactions or nanoscale mechanical movements. Herein, a nanoscale assembly line composing of dual DNA machines is meticulously designed by coupling a catalytic hairpin assembly (CHA)-based machine with a 3D DNA walker machine. Equipped with upconversion nanoparticles (UCNPs) as signal tags, the dual DNA machines-based assembly line (DDMAL) can efficiently amplify the fluorescent signal of target recognition event, enabling sensitive detection of microRNA (miRNA). In detail, once activated by target miRNA-21, the CHA machine is initiated to constantly produce a single-stranded DNA (named binding DNA) via the strand displacement reaction. The binding DNA as a trigger factor can initiate the DNA walker machine by linking a walking strand DNA with an anchor strand DNA immobilized on the surface of magnetic beads (MBs). The movement of walking strand on the surface of MBs is then driven by Mn2+-dependent DNAzyme formed through the hybridization of walking strand with a UCNPs-linked substrate strand. The DNAzyme-catalyzed cleavage of substrate strand is accompanied by the release of numerous UCNPs from MBs. By measuring the fluorescent signal of released UCNPs after the magnetic separation, target miRNA-21 can be detected by the DDMAL system in a linear range from 1.0 fM to 10 nM, with a limit of detection (LOD) of 0.62 fM (3σ). Moreover, the practicability of DDMAL system was demonstrated by using it to evaluate the expression levels of miRNA-21 in cell lines and assay miRNA-21 in human serum.

Development of a Pharmacogenetic Lab-on-Chip Assay Based on the In-Check Technology to Screen for Genetic Variations Associated to Adverse Drug Reactions to Common Chemotherapeutic Agents.

Background: Antineoplastic agents represent the most common class of drugs causing Adverse Drug Reactions (ADRs). Mutant alleles of genes coding for drug-metabolizing enzymes are the best studied individual risk factors for these ADRs. Although the correlation between genetic polymorphisms and ADRs is well-known, pharmacogenetic tests are limited to centralized laboratories with expensive or dedicated instrumentation used by specialized personnel. Nowadays, DNA chips have overcome the major limitations in terms of sensibility, specificity or small molecular detection, allowing the simultaneous detection of several genetic polymorphisms with time and costs-effective advantages. In this work, we describe the design of a novel silicon-based lab-on-chip assay able to perform low-density and high-resolution multi-assay analysis (amplification and hybridization reactions) on the In-Check platform. Methods: The novel lab-on-chip was used to screen 17 allelic variants of three genes associated with adverse reactions to common chemotherapeutic agents: DPYD (Dihydropyrimidine dehydrogenase), MTHFR (5,10-Methylenetetrahydrofolate reductase) and TPMT (Thiopurine S-methyltransferase). Results: Inter- and intra assay variability were performed to assess the specificity and sensibility of the chip. Linear regression was used to assess the optimal hybridization temperature set at 52 °C (R2 ≈ 0.97). Limit of detection was 50 nM. Conclusions: The high performance in terms of sensibility and specificity of this lab-on-chip supports its further translation to clinical diagnostics, where it may effectively promote precision medicine.

Gene-PROBER – a tool to design polynucleotide probes for targeting microbial genes

Recent developments in fluorescence in situ hybridization (FISH) methods allow the detection and visualization of the genes/genomic regions of bacteria, archaea and infecting viruses at the single cell level. These methods use mixtures of polynucleotides as probes to specifically detect the target of interest. Gene-PROBER enables the design of polynucleotide mixtures for targeting genes or genomic regions in microorganisms. It has four workflows, depending on the availability of non-target sequences and the choice of probe synthesis, either by chemical synthesis or by PCR. It outputs polynucleotides that are spread along the target sequence and have similar melting properties. Therefore, such a polynucleotide mixture can be used as a single probe, in a single hybridization reaction. Gene-PROBER is a freely available web service that can be accessed at http://gene-prober.icbm.de/, and is implemented in the R language using the Shiny package.

Glycerol Reduces Cross Hybridization on Nitrocellulose Membrane

Lateral flow assay (LFD) based nucleic acid lateral flow (NALF) method has been developed recently. The method met point of care testing (POCT) as simple and rapid procedures, less equipment, and can be performance by less skilled technician. NALF based on nucleic acid hybridizationis more economical then immunochromatography assay which use antibody-antigen recognition. Cross hybridization has issued while used to differentiate organism with high GC content and high homology as high similarity genome. Some techniques has applied to give high stringency condition avoid cross hybridization reaction but need more procedure to apply. We found glycerol applied to buffer assay could reduce cross hybridization on nitrocellulose membrane. The study used 2 kinds of high stringency buffer as PBS and SSC bases and high concentration of ssDNA amplicon as sample. Without glycerol ingredient gave cross hybridization signal on test line. But used glycerol could reduce those even omitted with PBS based buffer assay. Beside those, glycerol could significantly increased hybridization signal in SSC based buffer assay (p<0.05).

Complex Nucleic Acid Hybridization Reactions inside Capillary-Driven Microfluidic Chips.

Nucleic acid hybridization reactions play an important role in many (bio)chemical fields, for example, for the development of portable point-of-care diagnostics, and often such applications require nucleic acid-based reaction systems that ideally run without enzymes under isothermal conditions. The use of novel capillary-driven microfluidic chips to perform two isothermal nucleic acid hybridization reactions, the simple opening of molecular beacon structures and the complex reaction cascade of a clamped-hybridization chain reaction (C-HCR), is reported here. For this purpose, reagents are arranged in a self-coalescence module (SCM) of a passive silicon microfluidic chip using inkjet spotting. The SCM occupies a footprint of ≈7 mm2 of a ≈0.4×2cm2 microfluidic chip. By means of fluorophore-labeled DNA probes, the hybridization reactions can be analyzed in just ≈2min and using only ≈3µL of the sample. Furthermore, the SCM chip offers a variety of reagent delivery options, allowing, for example, the influence of the initiator concentration on the kinetics of C-HCR to be investigated systematically with minimal sample and time requirements. These results suggest that self-powered microfluidic chips equipped with a SCM provide a powerful platform for performing and investigating complex reaction systems.

Signal-off photoelectrochemical biosensing platform based on hybridization chain-doped manganese porphyrin quenching on CdSe signal coupling with cyclic amplification for thrombin detection

A novel signal-off photoelectrochemical (PEC) biosensing platform based on hybridization chain-doped manganese porphyrin quenching on PEC signal of CdSe quantum dots (QDs) coupling with cyclic amplification was developed for thrombin detection. Target thrombin firstly induced DNA enzyme-aided cyclic amplification reaction and produced abundant DNA I. Then DNA I hybridized with capture DNA on the TiO 2 /CdSe QDs modified electrode to form DNA bridge, which triggered a linear chain hybridization reaction with S4 and S5. Numerous manganese porphyrin (MnPP) were doped in the long double-stranded DNA chain, which hindered electron transfer and resulted in obvious decrease of QDs PEC signal, achieving a sensitive detection of thrombin. This PEC biosensor exhibits excellent performance with a wide linear range from 1.0 f. to 100.0 nM and detection limit of 0.331 fM, and the recovery value ranged from 95.5% to 103.5%. The results suggest that this PEC biosensor has high sensitivity and precision, showing great application prospect in microbiological analysis of biomolecules and clinical medicine. • A novel signal-off PEC biosensor based on DNA linear chain-doped MnPP quenching to QDs was reported. • CdSe QDs and TiO 2 complex displayed strong PEC signal. • A large amount of manganese porphyrin doped in linear DNA chain greatly amplified PEC signal change. • DNA S1 and S2 specifically bind to thrombin to form the pliers structure with high selectivity.

Natural antimicrobial compounds immobilised on silica microparticles as filtering materials: Impact on the metabolic activity and bacterial viability of waterborne microorganisms

The aim of this work was to assess the capability of filtering materials based on silica microparticles functionalised with essential oil components (EOCs) to remove waterborne bacteria from water, and to elucidate the mechanism of action of the inhibitory effect of the filtering materials on the metabolic activity and viability of the studied pathogens. Different silica microparticles (25, 50, 75, 200 or 375 μ m ) were functionalised with carvacrol, eugenol, thymol and vanillin to obtain filtering materials which removal capability was evaluated using distilled water inoculated with Escherichia coli, Helicobacter pylori, Legionella pneumophila or Pseudomonas aeruginosa (10 4 –10 7 cells/mL). Water samples were filtered through different layer thicknesses (0.5, 1 or 1.5 cm) of the filtering materials and the microbial load retained was determined by plate count. In addition, fluorescent viability staining, determination of cellular ATP content, direct viable count–fluorescent in situ hybridisation (DVC–FISH) and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) analyses were performed to prove the materials’ antimicrobial properties. The results exhibited that EOC-functionalised supports were capable of eliminating waterborne microorganisms from water with log reduction values falling within the 3–5 range, whereas the non-functionalised materials did not present relevant inhibitory capacity. The irreversible effect of the EOC-functionalised supports on the viability and metabolic activity of treated bacteria was confirmed by fluorescent staining (absence or red stained cells) and DVC–FISH (no elongated cells). Cellular ATP content was significantly reduced after filtering the inoculated water samples through the EOC-functionalised supports (cATP values below 10 pg/mL). Similarly, the concentration of viable bacteria determined by PMA-qPCR showed the inhibitory effect of the developed materials with negative quantification values for H. pylori and values of 7.98 ⋅ 10 1 –6.07 ⋅ 10 3 GU/mL for L. pneumophila water samples filtered with the EOC-functionalised supports. Thus, the use of the functionalised filtering materials led to loss of bacterial viability of the treated microorganisms with irreversible morphological and metabolic alterations, which confirms their potential use as filtering aids with additional properties for the biological control of water. • Filters based on essential oil components immobilised on silica supports were made. • The developed filters were used to remove and inactivate waterborne bacteria. • As proof of concept study, removal capacity fulfilled water quality requirements. • Viability assays confirmed the antimicrobial properties of filtering materials.

Reconfiguring DNA Nanotube Architectures via Selective Regulation of Terminating Structures.

Molecular assemblies inside cells often undergo structural reconfiguration in response to stimuli to alter their function. Adaptive reconfiguration of cytoskeletal networks, for example, enables cellular shape change, movement, and cargo transport and plays a key role in driving complex processes such as division and differentiation. The cellular cytoskeleton is a self-assembling polymer network composed of simple filaments, so reconfiguration often occurs through the rearrangement of its component filaments' connectivities. DNA nanotubes have emerged as promising building blocks for constructing programmable synthetic analogs of cytoskeletal networks. Nucleating seeds can control when and where nanotubes grow, and capping structures can bind nanotube ends to stop growth. Such seeding and capping structures, collectively called termini, can organize nanotubes into larger architectures. However, these structures cannot be selectively activated or inactivated in response to specific stimuli to rearrange nanotube architectures, a key property of cytoskeletal networks. Here, we demonstrate how selective regulation of the binding affinity of DNA nanotube termini for DNA nanotube monomers or nanotube ends can direct the reconfiguration of nanotube architectures. Using DNA hybridization and strand displacement reactions that specifically activate or inactivate four orthogonal nanotube termini, we demonstrate that nanotube architectures can be reconfigured by selective addition or removal of distinct termini. Finally, we show how terminus activation could be a sensitive detector and amplifier of a DNA sequence signal. These results could enable the development of adaptive and multifunctional materials or diagnostic tools.

P16 Immunoexpression in sinonasal and nasopharyngeal adenoid cystic carcinomas: a potential pitfall in ruling out HPV‐related multiphenotypic sinonasal carcinoma

Adenoid cystic carcinoma (AdCC) is frequent in the sinonasal region. The recently described human papilloma virus (HPV)-related multiphenotypic sinonasal carcinoma (HMSC) histopathologically resembles solid AdCC, but has a better outcome. Thus, clinical and pathogenetic differences between HMSC and sinonasal AdCC necessitate their distinction. We conducted this study to assess p16 immunoexpression in previously diagnosed AdCC cases, and to identify HMSC cases in p16 immunopositive cases. Cases diagnosed as sinonasal and nasopharyngeal AdCC were retrieved. Histomorphological features were reviewed. Immunohistochemistry (IHC) for p16 was performed. HPV testing was performed in p16-positive cases by mRNA in-situ hybridisation (mRNA ISH) and polymerase chain reaction (PCR) assay. MYB rearrangement was assessed by fluorescence in-situ hybridisation. One hundred and two AdCC cases were retrieved. Six cases (5.9%) showed diffuse p16 positivity. HPV mRNA ISH and PCR were negative in p16-positive cases. Two cases showed MYB rearrangement. p16-positive cases were composed of basaloid cells demonstrating a cribriform pattern, at least focally. The predominant pattern was cribriform in three and solid in three cases. One case showed two distinct components: keratinising squamous cell carcinoma and cribriform AdCC. Other morphological patterns seen were tubular, reticular, epithelial-myoepithelial carcinoma-like, and glomeruloid, forming a minor component of the tumour area. p16 staining alone, even when diffuse and strong, cannot be used as a surrogate for HPV testing to distinguish sinonasal AdCC from HMSC. p16 IHC should be accompanied by more specific methods, such as mRNA ISH, so as not to erroneously diagnose HMSC over sinonasal AdCC, bearing in mind the highly aggressive nature of the latter.

A Near‐Infrared Photo‐Switched MicroRNA Amplifier for Precise Photodynamic Therapy of Early‐Stage Cancers

AbstractStimuli‐responsive photodynamic therapy (PDT) is a hot topic in precise medicine, but the low abundance of responsive trigger molecules in early‐stage disease limits application. Here we designed an amplifier with multiple upconversion luminances to achieve a near‐infrared photo‐switched cascade reaction triggered by specific microRNA and precise PDT of early‐stage cancers. This amplifier was composed of photo‐caged DNA nanocombs and an upconversion nanoparticle (UCNP) sensitized with IRDye 800CW. The nanocomb was prepared by assembling a photozipper‐protected hairpin and two kinds of hybridizable hairpin probes on a DNA skeleton. Upon 808‐nm light irradiation, the produced UV light cleaved off the photozipper to induce microRNA‐responsive cascade hybridization reaction, activating the photosensitizers linked to different hairpins to generate reactive oxygen species (ROS) under the simultaneously emitted blue light for efficient PDT.

A Near-Infrared Photo-Switched MicroRNA Amplifier for Precise Photodynamic Therapy of Early-Stage Cancers.

Stimuli-responsive photodynamic therapy (PDT) is a hot topic in precise medicine, but the low abundance of responsive trigger molecules in early-stage disease limits application. Here we designed an amplifier with multiple upconversion luminances to achieve a near-infrared photo-switched cascade reaction triggered by specific microRNA and precise PDT of early-stage cancers. This amplifier was composed of photo-caged DNA nanocombs and an upconversion nanoparticle (UCNP) sensitized with IRDye 800CW. The nanocomb was prepared by assembling a photozipper-protected hairpin and two kinds of hybridizable hairpin probes on a DNA skeleton. Upon 808-nm light irradiation, the produced UV light cleaved off the photozipper to induce microRNA-responsive cascade hybridization reaction, activating the photosensitizers linked to different hairpins to generate reactive oxygen species (ROS) under the simultaneously emitted blue light for efficient PDT.

A colorimetric sensor for DNA detection: Combination of synergistic coupling catalysis and significant distinction in the dimensional structure of DNA

Herein, the simple and highly sensitive colorimetric sensor for DNA detection was constructed using the close correlation between DNA dimension and peroxidase activity of graphene oxide (GO)/AuNPs nanocomposite. One-dimensional and linear ss-DNA was hybridized into double helix DNA of three-dimensional space conformation, resulting in that catalytic activity of GO/AuNPs for the oxidation of enzyme substrate TMB significantly enhanced. Thus, DNA dimension transformation caused by hybridization is converted into color rendering events of TMB oxidation. The results show that the dimensional transformation of DNA can efficiently regulate the peroxidase activity of GO/AuNPs. Together with the effective amplifying for the color reaction of TMB by GO/AuNPs’ synergistic coupling catalysis at the nanometer interface, the colorimetric sensor is highly sensitive to DNA detection. The detection limit is calculated to be 8.8 × 10−12 M, which is about 1 ~ 2 orders of magnitude lower than that of other nanozyme colorimetric analysis. Besides, this analysis only requires one-step hybridization reaction, and the whole analysis is carried out in the homogeneous system of liquid phase, which is extremely easy to operate and conducive to the automatic sensing design. The detected results for serum samples declare that this sensor has great potential in the application of real life systems.

Magnetically controlled colorimetric aptasensor for chlorpyrifos based on copper-based metal-organic framework nanoparticles with peroxidase mimetic property.

The fabrication of a magnetically controlled colorimetric aptasensor for chlorpyrifos isreported. The aptasensor was fabricated by the attachment of the colorimetric labels onto the magnetic carrier due to the hybridization reaction between the complementary DNA and aptamer. Chlorpyrifos detection was realized by monitoring the color changes of the TMB/H2O2 solution before and after incubation of the aptasensor with chlorpyrifos via exposure to external magnetic force. The color change was monitored at 650nm by UV-Vis spectrophotometer. Under the optimal conditions, this magnetically controlled Cu-MOF-based aptasensor showed a detection limit of 4.4ng/mL with a linear range of 0-1250ng/mL. The colorimetric aptasensor displayed high selectivity for chlorpyrifos toward other interfering pesticides. The aptasensor was successfully applied for the spiked test of chlorpyrifos in fruits and vegetable samples with good recovery, which were in agreement with data obtained by GC-MS analysis. This magnetically controlled Cu-MOF-based sensing strategy not only leads to development of efficient and facile phase separation, but also expands the MOF's target scope from H2O2 or glucose to pesticides. Graphical abstract.

Direct Analysis of Rare Circulating Tumor Cells in Whole Blood Based on Their Controlled Capture and Release on Electrode Surface.

The development of a simple, sensitive, and effective method for the analysis of circulating tumor cells (CTCs) is essential for cancer diagnosis and metastasis prediction. In this work, we have proposed an enzyme-free electrochemical method for specific capture, sensitive quantification, and efficient release of CTCs. To achieve this, the specific interaction between CTCs and the corresponding aptamer designed to be located in the identification probe (IP) will unfold the hairpin structure of IP. Consequently, IP will initiate a hybridization reaction to produce a duplex, which will further trigger the hybridization chain reaction (HCR) process to form a composite product of CTCs and double-stranded DNA polymers. Therefore, a significantly amplified signal readout can be obtained. Moreover, the composite product can be brought to the electrode surface by tetrahedral DNA nanostructures to achieve the purpose of capturing and quantifying CTCs. More significantly, these captured CTCs can be controlled released without compromising cell viability via a simple strand displacement reaction. Taking the breast cancer cell MCF-7 as a representative, the newly developed approach led to an ultralow detection limit of 3 cells mL-1, which is superior to several studies previously reported. The current method has also been demonstrated to analyze CTCs in human whole blood and hence revealed a great potential in the future.

One-step and DNA amplification-free detection of Listeria monocytogenes in ham samples: Combining magnetic relaxation switching and DNA hybridization reaction

Early screening of L. monocytogenes in ready-to-eat food can prevent and control its harmful effects. In this study, we propose a highly sensitive magnetic DNA sensor based on nucleic acid hybridization reaction and magnetic signal readout. We design the L. monocytogenes specific probe1 and probe2 and label them on the 30 and 250 nm magnetic nanoparticles, respectively. The hybridization reaction between the magnetic probes and DNA of L. monocytogenes could form a sandwich nanocomplex. After magnetic separation, the unbound MNP30–probe2 can act as the transverse relaxation time (T2) signal readout probe. This assay allows the one-step detection of L. monocytogenes as low as 50 CFU/mL within 2 h without DNA amplification, and the average recovery in the spiked ham sausage samples can reach 92.6%. This system integrates the high sensitivity of magnetic sensing and high efficiency of hybridization reaction, providing a promising detection platform for pathogens.