Target DNA Molecules Research Articles

Spectral-Free Double Light Detection of DNA Based on a Porous Silicon Bragg Mirror.

To improve the detection sensitivity of a porous silicon optical biosensor in the real-time detection of biomolecules, a non-spectral porous silicon optical biosensor technology, based on dual-signal light detection, is proposed. Double-light detection is a combination of refractive index change detection and fluorescence change detection. It uses quantum dots to label probe molecules to detect target molecules. In the double-signal-light detection method, the first detection-signal light is the detection light that is reflected from the surface of the porous silicon Bragg mirror. The wavelength of the detection light is the same as the wavelength of the photonic band gap edge of the porous silicon Bragg mirror. CdSe/ZnS quantum dots are used to label the probe DNA and hybridize it with the target DNA molecules in the pores of porous silicon to improve its effective refractive index and enhance the detection-reflection light. The second detection-signal light is fluorescence, which is generated by the quantum dots in the reactant that are excited by light of a certain wavelength. The Bragg mirror structure further enhances the fluorescence signal. A digital microscope is used to simultaneously receive the digital image of two kinds of signal light superimposed on the surface of porous silicon, and the corresponding algorithm is used to calculate the change in the average grey value before and after the hybridization reaction to calculate the concentration of the DNA molecules. The detection limit of the DNA molecules was 0.42 pM. This method can not only detect target DNA by hybridization, but also detect antigen by immune reaction or parallel biochip detection for a porous silicon biosensor.

Conferring the Midas Touch on Integrative Taxonomy: A Nanogold-Oligonucleotide Conjugate-Based Quick Species Identification Tool

Nanogold or functionalized gold nanoparticles (GNPs) have myriad applications in medical sciences. GNPs are widely used in the area of nanodiagnostics and nanotherapeutics. Applications of GNPs in taxonomic studies have not been studied vis-à-vis its extensive medical applications. GNPs have great potential in the area of integrative taxonomy. We have realized that GNPs can be used to visually detect animal species based on molecular signatures. In this regard, we have synthesized gold nanoparticles (<20 nm) and have developed a method based on interactions between thiolated DNA oligonucleotides and small-sized GNPs, interactions between DNA oligonucleotides and target DNA molecules, and self-aggregating properties of small-sized GNPs under high salt concentrations leading to a visible change in colour. Exploiting these intermolecular and interparticle interactions under aqueous conditions, in the present work, we have demonstrated the application of our procedure by using a DNA oligonucleotide probe designed against a portion of the mitochondrial genome of the codling moth Cydia pomonella. This method is accurate, quick, and easy to use once devised and can be used as an additional tool along with DNA barcoding. This tool can be used for distinguishing cryptic species, identification of morphovariants of known species, diet analysis, and identification of pest species in quarantine facilities without any need of performing repetitive DNA sequencing. We suggest that designing and selecting a highly specific DNA probe is crucial in increasing the specificity of the procedure. Present work may be considered as an effort to introduce nanotechnology as a new discipline to the extensive field of integrative taxonomy with which disciplines like palaeontology, embryology, anatomy, ethology, ecology, biochemistry, and molecular biology are already associated for a long time.

Sensitive Electrochemical Biosensor for Rapid Screening of Tumor Biomarker TP53 Gene Mutation Hotspot.

Rapid and sensitive detection of cancer biomarkers is crucial for cancer screening, early detection, and improving patient survival rate. The present study proposes an electrochemical gene-sensor capable of detecting tumor related TP53 gene mutation hotspots by self-assembly of sulfhydryl ended hairpin DNA probes tagged with methylene blue (MB) onto a gold electrode. By performing a hybridization reaction with the target DNA sequence, the gene-sensor can rearrange the probe’s structure, resulting in significant electrochemical signal differences by differential pulse voltammetry. When the DNA biosensor is hybridized with 1 μM target DNA, the peak current response signal can decrease more than 60%, displaying high sensitivity and specificity for the TP53 gene. The biosensor achieved rapid and sensitive detection of the TP53 gene with a detection limit of 10 nmol L−1, and showed good specific recognition ability for single nucleotide polymorphism (SNP) and base sequence mismatches in the TP53 gene affecting residue 248 of the P53 protein. Moreover, the biosensor demonstrated good reproducibility, repeatability, operational stability, and anti-interference ability for target DNA molecule in the complex system of 50% fetal bovine serum. The proposed biosensor provides a powerful tool for the sensitive and specific detection of TP53 gene mutation hotspot sequences and could be used in clinical samples for early diagnosis and detection of cancer.

Real-time label-free assessment of T7 DNA polymerase immobilization

Immobilization of DNA-modifying enzymes on any surface is still a complex and challenging task in biotechnology and biosensorics. Therefore, this task very often is crucial, especially in biosensors dedicated to continuous monitoring of DNA. The novelty and the importance of this study are related to the development of continuously operating biosensors based on DNA-modifying enzymes. In this research we focused on the real-time monitoring of T7 DNA polymerase immobilization. Quartz crystal microbalance (QCM) was applied for the monitoring of immobilization of T7 DNA polymerase and assessment of analytical signals generated during the action of this enzyme. We have evaluated the covalent attachment of T7 DNA polymerase to different self-assembled monolayers consisting of different compositions of thiols, which were assembled on a gold-based surface of a QCM-resonator. To eliminate various aspects of inefficient T7 DNA polymerase immobilization and other inactivating factors associated with the inactivation of this DNA-enzyme, we have applied a strategy based on the protection of the exonuclease active site. We have examined the time-resolved simultaneous measurement of changes in frequency, energy dissipation, and surface saturation by T7 DNA polymerase during the immobilization process. Here reported findings suggest that detecting a low concentration of target DNA molecules can be achieved what enables efficient reuse of expensive DNA enzymes.

Comparison of Real-Time PCR and Droplet Digital PCR for the Quantitative Detection of Lactiplantibacillus plantarum subsp. plantarum

Droplet digital polymerase chain reaction (ddPCR) is one of the newest and most promising tools providing absolute quantification of target DNA molecules. Despite its emerging applications in microorganisms, few studies reported its use for detecting lactic acid bacteria. This study evaluated the applicability of a ddPCR assay targeting molecular genes obtained from in silico analysis for detecting Lactiplantibacillus plantarum subsp. plantarum, a bacterium mainly used as a starter or responsible for fermentation in food. The performance characteristics of a ddPCR were compared to those of a quantitative real-time PCR (qPCR). To compare the linearity and sensitivity of a qPCR and ddPCR, the calibration curve for a qPCR and the regression curve for a ddPCR were obtained using genomic DNA [102–108 colony-forming units (CFU)/mL] extracted from a pure culture and spiked food sample. Both the qPCR and ddPCR assays exhibited good linearity with a high coefficient of determination in the pure culture and spiked food sample (R2 ≥ 0.996). The ddPCR showed a 10-fold lower limit of detection, suggesting that a ddPCR is more sensitive than a qPCR. However, a ddPCR has limitations in the absolute quantitation of high bacterial concentrations (>106 CFU/mL). In conclusion, a ddPCR can be a reliable method for detecting and quantifying lactic acid bacteria in food.

A Simple and Sensitive Electrochemical DNA Biosensor of the Bacteria Chlamydia Trachomatis

Bacterial infection is a global problem, and detection of bacteria is the first step for solving such a problem. Herein, we developed an electrochemical biosensor for the detection of the bacteria Chlamydia Trachomatis. The hybridization-based biosensor was made by modifying the screen-printed gold electrode (SPGE) with the thiolated specific detection probes, which were complementary sequences to the target DNA molecule of the bacteria. The Oracet blue was used as an electrochemical label which was intercalated between two DNA sequences, and its reduction peak current was recorded by DPV method as an output signal of the biosensor. Conventional electrochemical characterization techniques, including cyclic voltammetry (CV) and Electrochemical impedance spectroscopy (EIS), were used to confirm the fabrication of the modified electrode. In addition, the Atomic Force Microscopy (AFM) imaging was performed to assess the electrode surface. The dynamic range of the biosensor was from 4 to 3000 pM with a detection limit of 1.3 pM. The simplicity and performance mentioned above of the biosensor, alongside the low cost and repeatability of the production, make it a great candidate for clinical applications for Chlamydia Trachomatis detection. Plus, it can be used for another species of bacteria with just a change of the thiolated probe.

Ultrasensitive Label-Free DNA Detection Based on Solution-Gated Graphene Transistors Functionalized with Carbon Quantum Dots.

Developing highly sensitive, reliable, cost-effective label-free DNA biosensors is challenging with traditional fluorescence, electrochemical, and other techniques. Most conventional methods require labeling fluorescence, enzymes, or other complex modification. Herein, we fabricate carbon quantum dot (CQD)-functionalized solution-gated graphene transistors for highly sensitive label-free DNA detection. The CQDs are immobilized on the surface of the gate electrode through mercaptoacetic acid with the thiol group. A single-stranded DNA (ssDNA) probe is immobilized on CQDs by strong π-π interactions. The ssDNA probe can hybridize with the ssDNA target and form double-stranded DNA, which led to a shift of Dirac voltage and the channel current response. The limit of detection can reach 1 aM which is 2-5 orders of magnitude lower than those of other methods reported previously. The sensor also exhibits a good linear range from 1 aM to 0.1 nM and has good specificity. It can effectively distinguish one-base mismatched target DNA. The response time is about 326 s for the 1 aM target DNA molecules. This work provides good perspectives on the applications in biosensors.

Retrospective Review and Scenario Analysis for qPCR Quality Assurance.

Quantitative polymerase chain reaction (qPCR) is a molecular method used in laboratories for detection and quantification of DNA nucleotides. Standard curves and reference values are usually combined into the PCR amplification process to calculate initial amount of target DNA molecule in a testing sample. This study is a retrospective review to examine the performance of standard curves in a qPCR-based assay for the screening of a genetic disorder in laboratories. Three different scenarios were designed to replay the historical data generated from over 3,000 qPCR assays to evaluate how omission of standard curves would affect the overall screening results. The outcomes of all the scenarios concluded that including standard curves in qPCR assays had decreased the screening specificity and accuracy, resulting in more false positives and additional retests. This was particularly true in case the initial DNA molecules were at relatively low copy numbers. It strongly suggests that the implantation of qPCR assays in diagnostic procedures should be frequently retested and reviewed. More importantly, the strategies of retrospective review and scenario analysis presented here will provide a good framework for assay validation and periodic quality assurance in laboratory.

Rapid detection of sweepoviruses through lateral flow dipstick-based recombinase polymerase amplification.

Sweepoviruses represent a phylogenetic group of begomoviruses that cause significant sweet potato (Ipomoea batatas) production losses in various countries across the world. For rapid identification of sweepoviruses, we developed a technique based on isothermal recombinase polymerase amplification in conjunction with lateral flow dipsticks (RPA-LFD). The optimum reaction conditions for the RPA were 20min incubation at 37°C. The RPA-LFD specifically detected distinct sweepovirus species, with no other viruses infecting sweet potato causing a cross-reaction. The detection limit of the RPA-LFD was 1.0×104 copies of the target DNA molecule per reaction, and it exhibited a 10-fold greater sensitivity than the conventional PCR. Furthermore, when coupled with an alkaline polyethylene glycol-based crude genomic DNA extraction, the entire procedure was completed in 30min without the use of any special instruments other than a water bath. Therefore, the RPA-LFD technique is a potential sweepovirus diagnostic tool that can be used in the field with fewer available resources. Keywords: detection; sweepoviruses; recombinase polymerase amplification; lateral flow dipstick.

A Review of DNA Data Storage Technologies Based on Biomolecules

: In the information age, data storage technology has become the key to improving computer systems. Since traditional storage technologies cannot meet the demand for massive storage, new DNA storage technology based on biomolecules attracts much attention. DNA storage refers to the technology that uses artificially synthesized deoxynucleotide chains to store and read all information, such as documents, pictures, and audio. First, data are encoded into binary number strings. Then, the four types of base, A(Adenine), T(Thymine), C(Cytosine), and G(Guanine), are used to encode the corresponding binary numbers so that the data can be used to construct the target DNA molecules in the form of deoxynucleotide chains. Subsequently, the corresponding DNA molecules are artificially synthesized, enabling the data to be stored within them. Compared with traditional storage systems, DNA storage has major advantages, such as high storage density, long duration, as well as low hardware cost, high access parallelism, and strong scalability, which satisfies the demands for big data storage. This manuscript first reviews the origin and development of DNA storage technology, then the storage principles, contents, and methods are introduced. Finally, the development of DNA storage technology is analyzed. From the initial research to the cutting edge of this field and beyond, the advantages, disadvantages, and practical applications of DNA storage technology require continuous exploration.

Open-Source Fluorescence Spectrometer for Noncontact Scientific Research and Education

Transforming fluorescence spectrometers into cost-effective, portable devices provides the potential for field-based applications in biological, environmental, and clinical research and education. However, the majority of developed spectroscopic technologies continue to require heavy, expensive equipment and trained personnel for operation or do not support multispectral analysis, thereby restricting their use in resource-limited environments. Herein, we report a wireless, portable, cost-effective, open-source fluorescence spectrometer (OpenFS) developed by compactly assembling optical and electronic elements in a 3D-printed housing. OpenFS outputs an accurate emission spectrum over a wide range of wavelengths and demonstrates greater sensitivity for fluorescence quantification compared to a conventional fluorometer. We demonstrate the functionality of OpenFS as a fluorescence resonance energy transfer (FRET)-based DNA sensor by detecting target DNA molecules with FRET efficiency and prove its utility as an Internet of Things device by performing wireless measurements and spectral analysis on a smartphone with a custom-developed Android application. This portable open-source spectrometer can lead to new opportunities in research and educational fields where fluorescence spectroscopy has not been available because of its cost and size and provides the potential for the development of mobile diagnostics platforms.

The CTPase activity of ParB determines the size and dynamics of prokaryotic DNA partition complexes

ParB-like CTPases mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromere of target DNA molecules and spread laterally to form large nucleoprotein complexes serving as docking points for the DNA segregation machinery. Here, we solve crystal structures of ParB in the pre- and post-hydrolysis state and illuminate the catalytic mechanism of nucleotide hydrolysis. Moreover, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. The study of CTPase-deficient ParB variants reveals that CTP hydrolysis serves to limit the sliding time of ParB clamps and thus drives the establishment of a well-defined ParB diffusion gradient across the centromere whose dynamics are critical for DNA segregation. These findings clarify the role of the ParB CTPase cycle in partition complex assembly and function and thus advance our understanding of this prototypic CTP-dependent molecular switch.

Coenzyme−catalyzed electroinitiated reversible addition fragmentation chain transfer polymerization for ultrasensitive electrochemical DNA detection

Ultrasensitive detection of biomarkers at an early stage is generally limited by external influence factors such as high reaction temperature, complex operations, and sophisticated instruments. Here, we circumvent these problems by using nicotinamide adenine dinucleotide (NAD+) to control electroinitiated reversible addition fragmentation chain transfer (electro−RAFT) polymerization for biosensing that enables the detection of a few molecules of target DNA. In this coenzyme−catalyzed electro−RAFT polymerization, numerous ferrocenylmethyl methacrylate (FCMMA) as monomer with electrochemistry signal were linked to the biomarker on Au electrode. Afterwards, a strong oxidation peak appears at the potential of about 0.3 V that represents a typical oxidation potential of FCMMA. The sensitivity of this methodology was presented by detecting DNA from 10−1 to 104 fM concentration and detection limit (LOD) being down to 4.39 aM in 10 μL samples. This is lower by factors than detection limits of most other ultra−sensitive electrochemical DNA assays.

Prdi-BF-1 Strongly Detected in Active Multiple Myeloma By Droplet Digital PCR: A Comparison with Real-Time PCR

Human positive regulatory domain I binding factor 1 (PRDI-BF1 or BLIMP-1) is a transcription factor that has been demonstrated to act as a master regulator required and sufficient for the generation and for the prolonged maintenance of plasma cells (PC). PRDI-BF1 essentially functions as a repressor, causing exit from cell cycling and the extinction of the expression of several genes critical for B-cell development at earlier stages. The PRDM1 gene, which codifies for PRDI-BF1, contains an alternative promoter capable of generating a PRDI-BF1 deleted protein (called PRDI-BF1β), which lacks 101 amino acids comprising most of the regulatory domain. The potential role of Blimp-1/PRDM1 in multiple myeloma, may help in understanding the progression of the disease and the potential specific function of a new drug as elotuzumab that selectively blocks CS1. To the best of our knowledge this is the first report that uses two new molecular techniques to identify and perform a quantitative levels of PRD-BF1. We measured, using real time polymerase chain reaction (RT- PCR) and Droplet digital PCR (ddPCR), the levels of PRDI-BF1 in Myeloma patients'(n=15) and normal human bone marrow (n=12). qPCR represents the most widely used method for molecular analysis. However, it has a major limitation from being a relative quantification approach. This results in the need of a reference standard curve usually built by dilutions of the tumor-specific target obtained from diagnostic DNA, plasmids, or cell lines that contain the rearrangement of interest. ddPCR is based on sample compartmentalization in single oil droplets that represent independent PCR reactions and on end point amplification and Poisson statistics. ddPCR has several theoretical advantages compared with qPCR. most notably allowing for absolute quantification of target DNA molecules and avoiding the need for a reference standard curve. dd-PCR was performed using the QX100 platform (BioRad®, Hercules, CA, USA), consisting of two instruments: the droplet generator and the droplet reader. The droplet generator divides the sample by creating about 20,000 partitions (droplets). The droplets are then transferred into PCR plates and, at the end of the amplification cycles, placed into the droplet reader, where each droplet is read as mutated or wild-type by issuing specific fluorescence signals (FAM for BLIMP and Hex for GAPDH). These signals, after being counted, are redistributed according to the Poisson's algorithm. In our samples, total RNA was extracted and reverse transcribed from bone marrow blood using RNA isolation and transcriptor kits from Roche (Barcelona, Spain). Primer mixes and TaqMan probes, assay references Hs00153357_m1 for PRDIBF1 and s99999903_m1 for β-actin (used as an internal refer-ence), were obtained from Applied Biosystems (Foster City, CA, USA). The relative quantitative values were calculated using the 2-∆∆CT method. In all MM patients the mean expression of PRDI-BF1 was 5.92 (range 1.00-21.75) fold higher than in healthy controls with RT PCR. Using ddPCR, PRDI-BF1 was also expressed at considerable levels in all samples tested and the mean expression of PRDI-BF1 was 10.10 (range 0.63-52.52) fold higher than in healthy controls.In particular in 4 patients with very aggressive disease the PRDI-BF had a marked higher transcript level (mean 28.52 range 15.12-52.52), Similar results were obtained with RT-PCR higher transcript level (mean 13.5 range 7.41-21.75) Our data suggest that the total level of PRDI-BF is clearly detectable in MM patients . The expression of these gene could be a feature for identifying active disease of MM with particular interest in very aggressive disease. DisclosuresGalimberti:Pfizer: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau; Incyte: Speakers Bureau.

Extraterritorial Patent Infringement: Gene Editing with CRISPR-Cas Perspective

The advent of CRISPR, with all its extraordinary potential, has exposed some seams and uncertainties in how U.S. patent law operates extraterritorially. The CRISPR-Cas system can be used to edit the human genome to correct diseases by way of a gRNA that guides a Cas enzyme to a certain DNA sequence location to make a corrective edit. Despite the presumption against extraterritorial patent protection, if an actor exports a component created in the U.S. that, when combined with other components abroad, will infringe a patented invention, the actor can be liable for infringement under 35 U.S.C. § 271(f). By way of an example, consider a patented invention that relates to a Cas9 protein and a DNA-targeting RNA with specific features (gRNA) to produce a modification of the targeted DNA molecule. If a party supplies a library of specific gRNAs for export to be combined with CRISPR-Cas9 in order to practice a patented invention as a whole, there may be 271(f) liability. Notably, the crux of the puzzle rests on the analysis of what a “component” is in such gene editing inventions. Courts could view Cas effector-encoding amino acid sequences, Cas protein domains, Cas effectors themselves, gRNA-encoding nucleotide sequences, exons, or gRNA molecules themselves as components. In this writing, I propose that sequences that encode the Cas effector proteins and the gRNAs be considered components for 271(f) liability purposes.

Cloning Polymerase Chain Reaction (PCR) Products: Blunt-End Cloning.

The following is an elegant and simple protocol for generating and cloning blunt-ended DNA. Incubation of a ligation reaction in the presence of an excess amount of restriction enzyme can dramatically increase the yield of recombinant plasmids. The role of the restriction enzyme is to cleave circular and linear concatemers at restriction sites that are regenerated when plasmid molecules ligate to themselves. The method requires that ligation of the plasmid to a target DNA molecule destroys the restriction site, so preventing the restriction enzyme from digesting recombinants generated during the ligation reaction. The net effect of constant reclamation of unit-length linear vector molecules is to drive the equilibrium of the ligation reaction strongly in favor of recombinants between vector and insert. The method is efficient because regeneration of vector DNA, ligation, and polishing the termini of PCR-generated fragments of DNA all occur simultaneously in the same reaction mixture.

A Biosensor Based on an Assembled Porous Silicon Microcavity

To solve the problem of the limited depth of penetration of biomolecules into silicon biosensors, which affects the performance of porous silicon devices, a new biological detection device assembled with porous silicon and Bragg structures is proposed that allows the biomolecules to directly enter the porous silicon microcavity layer, thus greatly shifting the resonance peak and improving the sensitivity of the porous silicon. The fabricated porous silicon devices with microcavities and Bragg structures are functionalized and coupled with biomolecules and then assembled with Bragg devices based on quartz glass to form the microcavity. Using the reflection spectrometer to detect the defect peak redshifts of different concentrations of target DNA molecules before and after reaction with the probe DNA molecules, obtaining the difference between the refractive indices before and after reaction. With an increase in the molecular concentration of the DNA in the target and an increase in the defect peak difference, the detection limit is 22.5 nM. The assembled microcavity has the advantages of high detection sensitivity, free labelling, low costs and so on.

DNA immobilization and detection using DNAbinding proteins.

The immobilization of sensing bioreceptors is a critical feature affecting the final performance of a biosensor. For DNA detection, the (strept)avidin-biotin affinity interaction is often used for the immobilization of biotin-labeled oligonucleotides or PCR amplicons. Herein, DNAbinding proteins are proposed as alternative universal anchors for both DNA immobilization and detection, based on the strong and specific affinity interaction between certain DNAbinding proteins and their respective dsDNA binding sites. These binding sites can be incorporated in the target DNA molecule during synthesis and by PCR, eliminating the need for post-synthesis chemical modification and resulting in lower costs. When scCro DNAbinding protein was immobilized on microplates and nitrocellulose membrane, both ssDNA and dsDNA targets were successfully detected. The detection limits achieved were similar to those obtained with the streptavidin-biotin system. However, the scCro system resulted in higher signals while using less amount of protein. The adsorption properties of scCro were superior to streptavidin's, making scCro a viable alternative as an anchor biomolecule for the development of DNA assays and biosensors. Finally, a nucleic acid lateral flow assay based solely on two different DNAbinding proteins, scCro and dHP, was developed for the detection of a PCR amplicon. Overall, the proposed system appears to be very promising and with potential use for multiplex detection using various DNAbinding proteins with different sequence specificities. Further work is required to better understand the adsorption properties of these biomolecules on nitrocellulose, optimize the assays comprehensively, and achieve improved sensitivities.

DNA nanotechnology provides an avenue for the construction of programmable dynamic molecular systems.

Self-assembled supramolecular structures in living cells and their dynamics underlie various cellular events, such as endocytosis, cell migration, intracellular transport, cell metabolism, and gene expression. Spatiotemporally regulated association/dissociation and generation/degradation of assembly components is one of the remarkable features of biological systems. The significant advancement in DNA nanotechnology over the last few decades has enabled the construction of various-shaped nanostructures via programmed self-assembly of sequence-designed oligonucleotides. These nanostructures can further be assembled into micrometer-sized structures, including ordered lattices, tubular structures, macromolecular droplets, and hydrogels. In addition to being a structural material, DNA is adopted to construct artificial molecular circuits capable of activating/inactivating or producing/decomposing target DNA molecules based on strand displacement or enzymatic reactions. In this review, we provide an overview of recent studies on artificially designed DNA-based self-assembled systems that exhibit dynamic features, such as association/dis­sociation of components, phase separation, stimulus responsivity, and DNA circuit-regulated structural formation. These biomacromolecule-based, bottom-up approaches for the construction of artificial molecular systems will not only throw light on bio-inspired nano/micro engineering, but also enable us to gain insights into how autonomy and adaptability of living systems can be realized.

Sequence-Specific Detection of DNA Strands Using a Solid-State Nanopore Assisted by Microbeads.

Simple, rapid, and low-cost detection of DNA with specific sequence is crucial for molecular diagnosis and therapy applications. In this research, the target DNA molecules are bonded to the streptavidin-coated microbeads, after hybridizing with biotinylated probes. A nanopore with a diameter significantly smaller than the microbeads is used to detect DNA molecules through the ionic pulse signals. Because the DNA molecules attached on the microbead should dissociate from the beads before completely passing through the pore, the signal duration time for the target DNA is two orders of magnitude longer than free DNA. Moreover, the high local concentration of target DNA molecules on the surface of microbeads leads to multiple DNA molecules translocating through the pore simultaneously, which generates pulse signals with amplitude much larger than single free DNA translocation events. Therefore, the DNA molecules with specific sequence can be easily identified by a nanopore sensor assisted by microbeads according to the ionic pulse signals.