Ultrasensitive DNA Detection Research Articles

Ultrasensitive electrochemiluminescence detection of DNA based on nanoporous gold electrode and PdCu@carbon nanocrystal composites as labels

A sensitive electrochemiluminescence (ECL) DNA biosensor based on nanoporous gold (NPG) electrode and PdCu@carbon nanocrystals (CNCs) composites is developed. The CNCs were obtained simply by electrooxidation with abundant carboxyl groups at their surfaces. The NPG can be easily prepared by a selective dissolution of silver from silver-gold alloy in nitric acid, which has free-standing noble metal membranes with controllable three-dimensional (3D) porosity. The PdCu bimetallic nanocomposites with hierarchically hollow structures were fabricated through a simple replacement reaction using dealloyed nanoporous copper (NPC) as both a template and reducing agent. Structure characterization was obtained by means of transmission electron microscope (TEM) and scanning electron microscope (SEM) images. The PdCu@CNCs composites exhibit 6 times higher ECL intensity than the pure CNC-labeled reporter DNA. Taking advantage of dual-amplification effects of the developed probe, a limit of detection as low as 18 aM can be achieved and the assay exhibits excellent selectivity for single-mismatched DNA detection even in human serum. The proposed ECL based method should have wide applications in diagnosis of genetic diseases due to its simplicity, low cost, and high sensitivity at extremely low concentrations.

An amplified single-walled carbon nanotube-mediated chemiluminescence turn-on sensing platform for ultrasensitive DNA detection

We have developed an amplified chemiluminescence turn-on sensing platform that relies on single-walled carbon nanotubes for ultrasensitive DNA detection. This new type of assay exhibits higher detection sensitivity over traditional biosensors by three orders of magnitude and high specificity for the target molecules.

Electrochemical growth of gold nanoparticles on horizontally aligned carbon nanotubes: A new platform for ultrasensitive DNA sensing

A new platform based on electrochemical growth of Au nanoparticles on horizontally aligned single walled carbon nanotube (SWCNT) array was developed for ultrasensitive DNA detection. The as-prepared DNA-functionalized SWCNT-Au platform, in which every gold-coated SWCNT acts as an isolated micro electrode, could detect lower than 10zmol complimentary 10-base DNA, which corresponded to having 6 DNA molecules in a 1mL sample solution. For a 1-base mismatched DNA, the experimental detection limit was 100amol. A linear relationship between the change of charge transfer resistance and target DNA concentration was achieved at low concentration range. Over the extended DNA concentration range, the change of charge transfer resistance was found to have a linear relationship with respect to the logarithm of the target DNA concentration. The sensor also showed great stability and could be conveniently regenerated via dehybridization in hot water. The significant improvement in sensitivity illustrates that combining Au nanoparticles with the on-site fabricated SWCNT array represents a promising platform for achieving ultrasensitive biosensor.

Effects of Chiral Gold Nanoflowers on Polymerase Chain Reaction

In this paper, we reported that chiral gold nanoflowers with the concentration of 0.08mMcan enhance the specificity of polymerase chain reaction (PCR) and eliminate un specific products, and the efficiency of PCR decline sin some extentand the melt temperature of PCR products also raises slightly, which may be associated with the enhancement of the combining power of primers and ssDNA templates and the inner stability of dsDNA by chiral gold nanoflowers. The phenomena have great potential in applications such as ultrasensitive specific detection of DNA, and SNPs.

Ultrasensitive DNA detection by cycle isothermal amplification based on nicking endonuclease and its application to logic gates

In recent years, an intense interest has grown in the DNA logic gates having high potential for computation at literally the “nano-size” level. A limitation of traditional DNA logic gates is that each target strand hybridizes with only a single copy of the probe. This 1:1 hybridization radio limits the gain of the approach and thus its sensitivity. The exponential amplification of nucleic acids has become a core technology in medical diagnostics and has been widely used for the construction of DNA sensor, DNA nanomachine and DNA sequencing. It would be of great interest to develop DNA-based logic systems with exponential amplification for the output signal. In the present study, a series of three-input DNA logic gates with the cycle isothermal amplification based on nicking endonuclease (NEase) are designed. Very low concentrations of the analytes were sufficient to initiate an autocatalytic cascade, achieving a significant improvement of the detection limit, 100-fold improvement compared to the non-autocatalytic system. This was achieved by engineering a simple and flexible biological circuit designed to initiate a cascade of events to detect and amplify a specific DNA sequence. This procedure has the potential to greatly simplify the logic operation because amplification can be performed in “one-pot”.

Combining a Nanowire SERRS Sensor and a Target Recycling Reaction for Ultrasensitive and Multiplex Identification of Pathogenic Fungi

Development of a rapid, sensitive, and multiplex pathogen DNA sensor enables early diagnosis and, subsequently, the proper treatment of infectious diseases, increasing the possibility to save the lives of infected patients. Here, the development of an ultrasensitive and multiplex pathogen DNA detection method that combines a patterned Au nanowire (NW)-on-film surface-enhanced resonance Raman scattering (SERRS) sensor with an exonuclease III-assisted target DNA recycling reaction is reported. Multiple probe DNAs are added to the target DNA solution, and among them, only the complementary probe DNA is selectively digested by exonuclease III, resulting in the decrease in its concentration. The digestion process is repeated by recycling of target DNAs. The decrease of the complementary probe DNA concentration is detected by SERRS. Combining the high sensitivity of the NW-on-film sensor and the target recycling reaction significantly improves DNA detection performance, resulting in the detection limit of 100 fM corresponding to 3 amole. By positioning Au NWs at specific addresses, multiple pathogen DNAs can be identified in a single step. Clinical sample tests with multiple genomic DNAs of pathogens show the potential of this sensor for practical diagnosis of infectious diseases.

Functionalized gold nanoparticles for ultrasensitive DNA detection

A major challenge in the area of DNA detection is the development of rapid methods that do not require polymerase chain reaction (PCR) amplification of the genetic sample. The PCR amplification step increases the cost of the assay, the complexity of the detection, and the quantity of DNA required for the assay. In this context, methods that are able to perform DNA analyses with ultrasensitivity have recently been investigated with the aim of developing new PCR-free detection protocols. Functionalized gold nanoparticles have played a central role in the development of such methods. Here, possibilities offered by functionalized gold nanoparticle in the ultrasensitive detection of DNA are discussed. The different functionalization protocols available for gold nanoparticles and the principal DNA detection methods that are able to detect DNA at the femtomolar to attomolar level are presented.

An integrated sensing system for detection of DNA using new parallel-motif DNA triplex system and graphene–mesoporous silica–gold nanoparticle hybrids

In this article, we demonstrate the use of graphene–mesoporous silica–gold NP hybrids (GSGHs) as an enhanced element of the integrated sensing platform for the ultra-sensitive and selective detection of DNA by using strand-displacement DNA polymerization and parallel-motif DNA triplex system as dual amplifications. We find that the present new sensing strategy based on GSGHs is able to detect target DNA with a fairly high detection sensitivity of 10 f m through the hybridization of duplex DNA to the acceptor DNA for the formation of parallel-motif DNA triplex on the multilayer film (containing GSGHs and redox probe) modified functional interface, and even has a good capability to investigate the single nucleotide polymorphisms (SNPs). The detection limit for target DNA is about two orders of magnitude lower than that of graphene-based DNA electrochemical impedance spectroscopy (EIS) sensor (6.6 p m), four orders of magnitude lower than those of graphene-based DNA sensors coupled with fluorescent assay (100 p m and 1 n m) and five orders of magnitude lower than those of field effect transistor (FET)-based assays (1 n m and 2 n m). Most importantly, our present sensing system can also be facilely achieved in the ITO electrode array, which is of paramount importance for possible multiplex analysis in lab-on-chip.

Magnetic particles and cadmium sulfide nanoparticles tagging for signal-amplifying detection of nucleic acids

A versatile DNA recognition system using cadmium sulfide nanoparticles (CdS NPs) as labels was proposed for ultrasensitive detection of specific sequence DNA based on target recycling. This strategy utilized the magnetic particles (MNPs) for the immobilization of linker DNA and CdS NPs and the subsequent target DNA hybridization. Using the unique characteristic of nicking endonuclease for cutting one specific strand of double strand DNA (ds DNA), the linker DNA could be transected and the target DNA could be liberated for re-hybridization and hence the amount of released CdS NPs was enhanced. Due to the advantage of the MNPs and signal amplification from the target recycling, the analyte DNA could be detected by the square-wave stripping voltammetry (SWV) in a wide linear range from 0.4 fM to 100 fM with the detection limit down to 0.08 fM. The proposed DNA detection strategy possesses high sensitivity, satisfactory reproducibility and excellent stability, which might have potential in other DNA biological assays.

Silicon nanowires-based highly-efficient SERS-active platform for ultrasensitive DNA detection

► A new silicon nanowires (SiNWs)-based SERS-active platform is developed for ultrahigh-sensitive DNA detection. ► This SiNWs-based platform possesses an exceptionally high SERS enhancement factor (∼10 10 ), which is developed to a high-performance DNA biosensor. ► This silicon-based biosensor can readily detect a remarkably low DNA concentration at 1 fM, which is the lowest DNA concentration ever detected via SERS. A nanostructured complex, silicon nanowires (SiNWs) coated with in situ grown AgNPs (SiNWs@AgNPs), is developed as a well-defined surface-enhanced Raman Scattering (SERS)-active platform for ultrasensitive DNA detection. Such SiNWs@AgNPs nanostructure possesses an extremely high SERS enhancement factor of ∼10 10 . We design a DNA sensor based on the nanostructure via immobilization of capture probe DNA at the surface of AgNPs, and a sandwich strategy for the detection of target DNA that brings a reporter probe labeled with a dye to the proximity of the surface, leading to high SERS signals. This silicon nanostructure-based biosensor platform can detect a remarkably low DNA concentration at ∼1 fM, which is comparable to the lowest DNA concentration ever detected via SERS. We expect this highly sensitive and robust SiNWs-based SERS platform may serve as a practical and powerful tool for biomolecular sensing and biomedical analysis.

Ultrasensitive DNA detection based on Au nanoparticles and isothermal circular double-assisted electrochemiluminescence signal amplification

We report here an ultrasensitive DNA detection approach which combines Au NPs enhanced electrochemiluminescence (ECL) of the CdS nanocrystal (NC) film with isothermal circular amplification reaction of polymerase and nicking endonuclease (NEase). By the double-signal amplification, this approach could sensitively respond down to 5 aM DNA.

Ultrasensitive detection of genomic DNA from apple leaf tissues, using surface-enhanced Raman scattering

Ultrasensitive detection of nucleic acids is demonstrated through exploiting the effect of surface-enhanced Raman scattering (SERS). In this work the SERS spectra of eight genomic DNAs from leaves of apple trees grown in the field (Malus domesticaBorkh., Fam.Rosaceae, cultivars. Florina, Idared, Rebra, Goldrush, Romus 3, Romus 4 and the rootstocks M9 and M26) have been analyzed in the wavenumber range 200–1800 cm–1. SERS signatures, spectroscopic band assignments and structural interpretations of these plant genomic DNAs are reported. SERS spectra of nucleic acids are compared here with caution, because these signals are time-dependent and are strongly influenced by DNA amount in the measured sample volume. Similarities of the SERS spectra of genomic DNAs extracted from apple leaves of Goldrush, Rebra and Florina cultivars, respectively, have been detected. Besides, the SERS spectra, corresponding to DNA from M9, Romus 4 and M26, leaves, respectively, show similar features and well resolved bands. Based on this work, specific plant DNA-ligand interactions or DNA structural changes induced by plant stress conditions associated with their natural environment, might be further investigated using surface-enhanced Raman spectroscopy.

Ultrasensitive detection of genomic DNA from apple leaf tissues, using surface-enhanced Raman scattering

Ultrasensitive detection of nucleic acids is demonstrated through exploiting the effect of surface-enhanced Raman scattering (SERS). In this work the SERS spectra of eight genomic DNAs from leaves of apple trees grown in the field (Malus domestica Borkh., Fam. Rosaceae, cultivars. Florina, Idared, Rebra, Goldrush, Romus 3, Romus 4 and the rootstocks M9 and M26) have been analyzed in the wavenumber range 200–1800 cm–1. SERS signatures, spectroscopic band assignments and structural interpretations of these plant genomic DNAs are reported. SERS spectra of nucleic acids are compared here with caution, because these signals are time-dependent and are strongly influenced by DNA amount in the measured sample volume. Similarities of the SERS spectra of genomic DNAs extracted from apple leaves of Goldrush, Rebra and Florina cultivars, respectively, have been detected. Besides, the SERS spectra, corresponding to DNA from M9, Romus 4 and M26, leaves, respectively, show similar features and well resolved bands. Based on this work, specific plant DNA-ligand interactions or DNA structural changes induced by plant stress conditions associated with their natural environment, might be further investigated using surface-enhanced Raman spectroscopy.

Ultrasensitive fluorescence polarization DNA detection by target assisted exonuclease III-catalyzed signal amplification

Single stranded DNA sequences can be detected by target assisted exonuclease III-catalyzed signal amplification fluorescence polarization (TAECA-FP). The method offers an impressive detection limit of 83 aM within one hour for DNA detection and exhibits high discrimination ability even against a single base mismatch.

Ultrasensitive and selective DNA detection by hydroxylamine assisted gold nanoparticle amplification

A new nanoparticle-based chemiluminescent (CL) method has been developed for the ultrasensitive detection of DNA hybridization which can achieve ultra-sensitivity up to approximately 30 zmol, i.e. 300 aM.

Ultrasensitive DNA Detection by Cascade Enzymatic Signal Amplification Based on Afu Flap Endonuclease Coupled with Nicking Endonuclease

Empfindliches Diagnosewerkzeug: Eine kaskadische enzymatische Signalverstärkung (CESA) für den DNA-Nachweis wird in drei Stufen erreicht: invasive Signalverstärkung durch Afu-Endonuklease zur Erzeugung amplifizierter Flap-Sequenzen, Ligation der Flap-Sequenzen durch T4-Ligase zur Bildung einer Nicking-Stelle und Nicking-Reaktion durch die Nicking-Endonuklease zur Signalverstärkung (siehe Bild für den letzten Schritt). CESA erreicht eine Empfindlichkeit von 1 fM DNA.

Ultrasensitive DNA Detection by Cascade Enzymatic Signal Amplification Based on Afu Flap Endonuclease Coupled with Nicking Endonuclease

Ultrasensitive DNA Detection by Cascade Enzymatic Signal Amplification Based on Afu Flap Endonuclease Coupled with Nicking Endonuclease

Ultrasensitive quantum dots-based DNA detection and hybridization kinetics analysis with evanescent wave biosensing platform

Ultrasensitive DNA detection was achieved using a new biosensing platform based on quantum dots (QDs) and total internal reflection fluorescence, which featured an exceptional detection limit of 3.2 amol of bound target DNA. The reusable sensor surface was produced by covalently immobilizing streptavidin onto a self-assembled alkanethiol monolayer of fiber optic probe through a heterobifunctional reagent. Streptavidin served as a versatile binding element for biotinylated single-strand DNA (ssDNA). The ssDNA-coated fiber probe was evaluated as a nucleic acid biosensor through a DNA–DNA hybridization assay for a 30-mer ssDNA, which were the segments of the uidA gene of Escherichia coli and labeled by QDs using avidin–biotin interaction. Several negative control tests revealed the absence of significant non-specific binding. It also showed that bound target DNA could easily be eluted from the sensor surface using SDS solution (pH 1.9) without any significant loss of performance after more than 30 assay cycles. A quantitative measurement of DNA binding kinetics was achieved with high accuracy, indicating an association rate of 1.38 × 10 6 M −1 s −1 and a dissociation rate of 4.67 × 10 −3 s −1. The proposed biosensing platform provides a simple, cheap, fast, and robust solution for many potential applications including clinical diagnosis, pathology, and genetics.

A Fluorogenic Reaction Based on Heavy-Atom Removal for Ultrasensitive DNA Detection

Fluorogenic reactions have recently emerged as a powerful tool for detection, diagnostics, and biosensing applications in a chemical and biological context. However, conventional fluorogenic systems reported to date rely on energy- or photoinduced electron transfer within the probes. Our communication demonstrates a conceptually new approach for generating a strong fluorescence signal through chemical bond formation mediated by a heavy-atom removal process. This method has favorable photophysical properties such as exceptional quantum yield and very low limits of fluorogenic DNA detection.

Two‐Photon Excited Fluorescence Enhancement for Ultrasensitive DNA Detection on Large‐Area Gold Nanopatterns

Ultrasensitive DNA detection using enhanced two-photon excited fluorescence: DNA detection systems formed on a highly regular gold nanopattern over a large area show enhanced sensitivity from high to ultralow concentrations (similar to 10-11 M, see figure) target molecules. The observed two-photon excited fluorescence on the gold patterned surface is enhanced over two orders of magnitude compared with the molecules on the glass surface.