Label-free DNA Detection Research Articles

A Graphene Oxide–Rhodamine 6G Nanocomposite as Turn-on Fluorescence Probe for Selective Detection of DNA

Abstract An electrostatic nanocomposite from Rhodamine 6G (R6G) and GO for the cost-effective, label-free, facile, sensitive and selective detection of DNA was constructed. Upon the interaction between positively charged dye and negatively charged GO, the charge-transfer complex R6G+GO- formed and significantly fluorescence quenching was observed. Due to the high electrostatic and non-covalent interactions between R6G and DNA, in the present of DNA, the interaction between R6G and GO was disturbed, which will turn on the fluorescence of R6G. The key advance of this R6G+GO- biosensor platform is attributed to the high quenching and absorbing capabilities of GO, which make it an effective candidate for DNA detection.

Optically amplified DNA detection on self-assembled solid films using conjugated polyelectrolytes

A new label-free heterogeneous DNA detection method was developed using the fluorescent conjugated polyelectrolyte poly[9,9′-bis(6′′-(N,N,N-trimethylammonium)-hexyl)fluorene-2,7-ylenevinylene-co-alt-1,4-phenylene dibromide] (PFV). In this assay, Layer-by-Layer (LbL) assembly was used to form a polyelectrolyte multilayer thin film on a substrate, and enhance the adsorption ability and stability of PFV. A cationic poly-(allylamine hydrochloride) (PAH) buffer layer was deposited to increase deposition of DNA. DNA detection was studied using a platform with the double-stranded DNA (dsDNA) intercalator ethidium bromide (EB). The thickness of the PAH layer was controlled by pH to obtain an optimum distance between PFV and EB for efficient fluorescence resonance energy transfer (FRET). Because of the different interactions of dsDNA and single-stranded DNA (ssDNA) with EB, DNA hybridization was selectively detected by monitoring the FRET efficiency.

Exonuclease-aided amplification for label-free and fluorescence turn-on DNA detection based on aggregation-induced quenching

A facile, universal and label-free fluorescence turn-on amplification strategy for the detection of DNA has been reported, which can achieve ultralow femtomolar detection of a target gene sequence with high selectivity.

Exonuclease-assisted cascaded recycling amplification for label-free detection ofDNA

The network consisting of three kinds of unlabeled stem-loop DNA molecular beacons (MBs) is activated by target DNA in the presence of exonuclease-III (Exo-III), achieving the concept of exonuclease-assisted cascaded recycling amplification (Exo-CRA) for DNA detection with a wide dynamic range of 8 orders of magnitude.

A low-cost, label-free DNA detection method in lab-on-chip format based on electrohydrodynamic instabilities, with application to long-range PCR

In order to evolve from a "chip in the lab" to a "lab on a chip" paradigm, there is still a strong demand for low-cost, portable detection technologies, notably for analytes at low concentrations. Here we report a new label-free DNA detection method with direct electronic read, and apply it to long-range PCR. This method uses a nonlinear electrohydrodynamic phenomenon: when subjected to high electric fields (typically above 100 V cm(-1)), suspensions of large polyelectrolytes, such as long DNA molecules, create "giant" dynamic concentration fluctuations. These fluctuations are associated with large conductivity inhomogeneities, and we use here a contact-mode local conductivity detector to detect these fluctuations. In order to decouple the detection electronics from the high voltage excitation one, an original "doubly symmetric" floating mode battery-operated detection scheme was developed. A wavelet analysis was then applied, to unravel from the chaotic character of the electohydrodynamic instabilities a scalar signal robustly reflecting the amplification of DNA. As a first proof of concept, we measured the products of the off-chip amplification of 10 kbp DNA from lambda phage DNA, achieving a sensitivity better than 100 fg DNA in the original 50 μl sample. This corresponds to the amplification products of less than 100 initial copies of target DNA. The companion enabling technologies developed to implement this new concept, i.e. the doubly symmetric contact conductivity detection and wavelet analysis, may also find various other applications in lab-on-chips.

Detection of DNA based on fluorescence resonance energy transfer of polyelectrolyte-protected CdTe quantum dots as energy donors

The approach for DNA detection was established by using a fluorescence resonance energy transfer (FRET) system, in which the energy donor was poly-diallyldimethylammonium chloride-protected quantum dots and the energy receptor was ethidium bromide (EB) inserting into the double stranded DNA. The concentration of the probe DNA, EB and NaCl was optimized. Under the optimized conditions, the FRET system has a stable signal and good reproducibility. The linear range is 7.7-61.6 nM with the correlation coefficient of 0.998 and the limit of detection is 7.7 nM. This method is simple and sensitive, and makes the label-free DNA detection come true.

Hierarchical optical antenna: Gold nanoparticle-modified photonic crystal for highly-sensitive label-free DNA detection

Metallic nanoparticles combined with 3D photonic crystals (PC) are anticipated to achieve high light-use efficiency, which stimulates their promising applications as optical antenna. Here, we propose a hierarchical optical antenna from an inverse opal photonic crystal modified with gold nanoparticles (GNPs) and exploit it for highly-sensitive bio-sensing. Metallic nanostructures can induce efficient light-harvesting and boost the light–matter interaction through localized surface plasmon resonance (LSPR). Photonic crystals are also efficient optical antennas with the ability to manipulate incident light. The 3D photonic crystal can localize the excitation energy and induce intense LSPR of GNPs for fluorescent enhancement when the PC band edge agrees with the LSPR absorption wavelength of the GNPs and the excitation wavelength of the light source. The feasibility of our approach is demonstrated by label-free DNA detection of SARS and HIV, and the GNP-modified inverse opal gives rise to one magnitude enhancement of the signal intensity and the detection sensitivity. This hierarchical antenna is an interesting prospect for highly effective light harvesting through the optimized combination of the superior properties of photonic crystals and plasmonic materials.

Label-Free Detection of DNA by Field-Effect Devices

DNA sensor and microarray technologies offer powerful tools for addressing various questions in biological and medical science. When these sensors and microarrays operate in a label-dependent manner, a low concentration of nucleic acid targets can be detected with excellent sensitivity. Nonetheless, label-free detection techniques eliminate labeling steps and therefore may allow rapid and low-cost assays. Field-effect devices provide a promising label-free approach for the high-throughput real-time monitoring of nucleic acids. This paper reviews major concepts and recent progress on the application of field-effect devices for DNA sensing. First, device architectures are described in terms of what events are detectable, how sensor systems are constructed, and how capture probes are immobilized on a gate surface. Hybridization detection, the limit of charge detection, and ways to overcome this limitation are also discussed.

Ordered silicon nanocones arrays for label-free DNA quantitative analysis by surface-enhanced Raman spectroscopy

Ordered vertical silicon nanocones arrays coated with silver nanoparticles (AgNPs@SiNCs) are developed as surface-enhanced Raman scattering (SERS)-active substrate, which features good uniformity and reliable reproducibility of SERS signals. Label-free DNA at low concentrations (10−8 M) could be quantitatively analyzed via SERS using the AgNPs@SiNCs. The Raman peak at 732 cm−1 due to adenine breathing mode was selected as an endogenous Raman marker for quantitative detection of label-free DNA. The AgNPs@SiNCs as high-performance SERS-active substrates are attractive for surface enhancement mechanism investigation and biochemical sensing applications.

Magnetic Bead-Based Multiplex DNA Sequence Detection of Genetically Modified Organisms Using Quantum Dot-Encoded Silicon Dioxide Nanoparticles

In this study, we report the multiplex DNA detection of genetically modified organisms (GMOs) based on fluorescence and magnetic nanostructures built up with quantum dot-encoded silicon dioxide nanoparticles (QD-encoded SiO2 NPs) and magnetic nanoparticles (MNPs). Detection was achieved by fluorescence spectrophotometry without auxiliary instruments. GMOs, including corn, soybean, colza, and cotton, were simultaneously detected, and the quantitative detection of soybean with a limit of detection of low femtomolar concentrations was successfully achieved. SiO2 NPs were encoded by different colored QDs at various ratios through a reverse microemulsion. QD-encoded SiO2 NP-labeled probe DNAs and MNP-labeled capture DNAs were used to hybridize with the corresponding targets at the same time. After magnetic separation, the sandwich structures were measured by fluorescence spectrophotometry. According to the overlay spectra of QDs, we estimated the presence of corresponding targets by analyzing the results via X-ray photoelectron spectroscopy peak software. Multiplex label-free DNA detection of GMOs based on QD-encoded SiO2 NPs showed high sensitivity and can be applied in other samples.

Functional magnetic nanoparticle-based trapping and sensing approaches for label-free fluorescence detection of DNA

In this study, a label-free fluorescence detection method for DNA was designed using functional magnetic nanoparticles (MNPs) as affinity probes. With the advantage of magnetic feature, MNP-based affinity probes can be easily manipulated for trapping and sensing target species. Two types of MNP-based nanoprobes for trapping and detecting target DNAs were fabricated. The basic strategy for this approach is the use of trapping probes to concentrate target DNAs selectively from complex samples. The detection probes are then used as fluorescence reporters to explore the level of the target species. Trapping probes were constructed by covalently immobilizing probe DNA molecules complementary to the target DNA. Detection nanoprobes were made by linking a fluorescent dye, riboflavin-5′-monophosphate (RFMP), onto the surface of the core/shell Fe3O4@Al2O3 MNPs via Al-phosphate chelation. The fluorescence derived from RFMP molecules became invisible when molecules were attached onto the MNP surface. However, after phosphorylated species (e.g., DNA molecules) replaced RFMP from the surface of the RFMP-Fe3O4@Al2O3 MNPs under microwave heating for 15s, the RFMP molecules released from the MNPs enhanced the fluorescence intensity in the solution. Based on the measurement of the fluorescence intensity, the level of target DNA in the samples was determined. The remaining DNA molecules on the RFMP-Fe3O4@Al2O3 MNPs were characterized by using matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The detection limit for DNA was as low as 40pM using this approach.

Elaboration of an Ag°/TiO2 platform for DNA detection by surface enhanced Raman spectroscopy

The promising potential of a new platform for the label-free DNA detection by Surface Enhanced Raman Spectroscopy (SERS) is reported. This platform is composed of silver nanoparticles distributed over the surface of a TiO2 (anatase) film. DNA molecules are immobilized on the platform using a simple grafting procedure. The elaboration and biomodification of the platform involve wet chemistry routes and benefit from specific properties of its components. First, the photocatalytic activity of TiO2 films is used for the photoreduction of Ag salts leading to the Ag° nanoparticle deposition. Second, the grafting and surface distribution of DNA benefit from both the photo-induced hydrophilicity of TiO2 films and the chemical affinity between metallic silver and amine ligands. Despite a broad size distribution of the Ag° nanoparticles which affects the reproducibility of DNA Raman spectra, specific changes of DNA Raman line intensities observed in single strand and hybridized DNA attest that hybridization can reliably be detected and that a spatially addressed DNA detection is possible.

Label free DNA detection based on gold nanoparticles quenching fluorescence of Rhodamine B

A novel and sensitive label free DNA detection method using gold nanoparticles (GNPs) and Rhodamine B (RB) has been developed. The assay is based on the following two properties. One is the different adsorption properties of single-stranded and double-stranded DNA on GNPs in colloidal solution. The other is the different quenching ability of aggregated GNPs and dispersed GNPs on RB. Un-aggregated GNPs could effectively quench the fluorescence of RB. However, the quenching ability greatly decreases after GNPs aggregated. The hybridization of probe DNA and target DNA is monitored by the fluorescence detection after the RB is added to the solution. Under the optimal experimental conditions, the detection limit of this assay is 2.9×10−13molL−1.

High sensitive and label-free colorimetric DNA detection based on nicking endonuclease-assisted activation of DNAzymes

Horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme) attracts growing interest as an amplifying label for biorecognition and biosensing events, especially for DNA detection. However, in the traditional designs, one target molecule can only generate one HRP-DNAzyme, which limits the signal enhancement and thus its sensitivity. In this article, we propose an amplified and label-free colorimetric DNA detection strategy based on nicking endonuclease (NEase)-assisted activation of HRP-DNAzymes (NEAA-DNAzymes). This new strategy relies on the hairpin-DNAzyme probe and NEase-assisted target recycling. In the hairpin-DNAzyme probe, the HRP-DNAzyme sequence is protected in a “caged” inactive structure, whereas the loop region includes the target complementary sequence. Upon hybridization with target, the beacon is opened, resulting in the activation of the HRP-DNAzyme. Meanwhile, upon formation of the duplex, the NEase recognizes a specific nucleotide sequence and cleaves the hairpin-DNAzyme probe into two fragments. After nicking, the fragments of the hairpin-DNAzyme probe spontaneously dissociate from the target DNA. Amplification is accomplished by another hairpin-DNAzyme probe hybridizing to the released intact target to continue the strand-scission cycle, which results in activation of numerous DNAzymes. The activated HRP-DNAzymes generate colorimetric or chemiluminescence readout signals, thus providing the amplified detection of DNA. The detection limit of the colorimetric method is 10pmol/L, which are three orders of magnitude lower than that without NEase. In addition, the detection limit of the chemiluminescence method is 0.2pmol/L. Meanwhile, this strategy also exhibits high discrimination ability even against single-base mismatch.

Label-free DNA detection with a nanogap embedded complementary metal oxidesemiconductor

A nanogap embedded complementary metal oxide semiconductor (NeCMOS) isdemonstrated as a proof-of-concept for label-free detection of DNA sequence. When apartially carved nanogap between a gate and a silicon channel is filled with chargedbiomolecules, the gate dielectric constant and charges are changed. When the gate oxidethickness reduces, the threshold voltage is significantly affected by a change of the charges,whereas it is scarcely influenced by a change of the dielectric constant. In the case of DNA,those two factors act on the threshold voltage oppositely in an n-channel NeCMOS butcollaboratively in a p-channel NeCMOS because of the negative charges of DNA. Hence, ap-channel NeCMOS with a thin gate oxide is more attractive for DNA detection because itenhances the shift of threshold voltage; that is, it improves the sensitivity of DNAdetection. In addition, the shift of threshold voltage according to the nanogap lengthis also investigated and the longer nanogap shows more shift of the thresholdvoltage.

Nanoscale Repulsive and Attractive Forces on Transistors - a Study of DNA Interaction with Non Volatile Charge

Label-free DNA detection by transistors is shown to have a tremendous potential to detect hybridization events and dielectric properties of adsorbed membranes within minutes. We report on an integrated-circuit detection scheme to monitor DNA-DNA interaction with high temporal resolution and novel features of manipulating the adsorbed DNA through long range in situ repulsive electrostatic forces. The floating-gate MOS transistor [1] can also be used as a detector in impedimetric mode by applying independent dc and ac biases from the control gate and the solution, respectively. This technique is shown to accurately measure changes in capacitance at the DNA-transistor interface as the operating point is held constant through the control gate. The output is a strong function of frequency. The transient IV and impedance spectroscopy then probe the surface adsorption, hybridization and molecular make up of the target DNA. In addition by using the property of fowler nordheim (FN) tunneling we can tunnel charges in and out of a floating gate. The stored charge creates an in situ refreshable mechanism at the interface that leads to DNA desorption. Experiments are underway to demonstrate addressable sensor pixel arrays. [1]. Krishna Jayant, Shantanu R. Rajwade, Lois Pollack and Edwin C.Kan, “Controlled Adsorption and Desorption on CMOS- Towards a Bi-Directional Bioelectronic Interface”, Biosensors (2010), World Congress on Biosensors and Bioelectronics, Glasgow, Scotland, UK 26-28thMay 2010. P31.081

Microcantilever Biosensors: Probing Biomolecular Interactions at the Nanoscale

Microcantilever based biosensors have attracted much attention as a label-free, real-time and highly sensitive approach to the detection of biomolecules. When specific biomolecular interactions occur between a receptor immobilized on one side of a cantilever and a target analyte in solution, a mechanical bending of the cantilever results due to a change in surface stress, thereby translating biochemical interactions into a concentration-dependent nanomechanical response of the microcantilevers. In this review, we present a general overview of the operation principles, fabrication and preparation of microcantilever biosensors and discuss their uses for a wide range of biosensing applications. The focus of the review is given to probing the biomolecular interactions at the solid-liquid interface by measuring the cantilever bending. In addition to the label-free detection of DNA, protein and cells, the emphasis is also made to discuss about the new development of microcantilever biosensors for label-free probing nanoscale conformational changes of DNA, protein and polymers, and real-time detection of nanomechanical forces from living cells. Finally, the outlook for future development work and challenges is also discussed. Keywords: Microcantilever biosensors, molecular interactions, conformational changes, microarrays, ELISAs, atomic force microscopy (AFM), label-free biosensor, microfabrication, Device Fabrication, AFM, Surface Functionalization, BIOSENSING APPLICATIONS, DNA hybridization reaction, single nucleo-tide polymorphisms (SNPs),, prostate-specific antigen (PSA), human serum albumin, human plasminogen, CDK2, drug vancomycin, mucopetide analogues, piezoelectric cantilever

Highly sensitive polysilicon wire sensor for DNA detection using silica nanoparticles/γ-APTES nanocomposite for surface modification

Poly-silicon wire (PSW) coated with a nanocomposite composed of a mixture of 3-aminopropyltriethoxysilane (γ-APTES) and polydimethylsiloxane (PDMS)-treated hydrophobic fumed silica nanoparticles (NPs) plus UV light illumination (γ-APTES + NPs + UV) is proposed for label-free DNA detection. We found that the linear detection range extended from 1 fM to 10 μM, with a lowest detection limit of 0.3 fM, could be achieved using the PSW sensor for the analysis of 10-base-long synthetic DNA including the single-strained (ss)-homopolymers poly-adenine [poly(A)], poly-thymine [poly-(T)], poly-cytosine [poly(C)], and poly-guanines [poly(G)]. The proposed PSW sensor can detect a single base change within a trace amount of a 5-base-long DNA molecule solution with an ultra-low concentration of 10 fM, which is loaded and transferred by the focus-ion-beam (FIB) milled AFM tip. The adsorption of DNA molecules onto the γ-APTES + NPs + UV surface is extremely efficient and follows the Langmuir adsorption isotherm. Fourier transform infrared (FTIR) spectroscopy analysis showed the improvement was attributable to the ethoxy groups in the γ-APTES that reacted chemically with the silica NPs surfaces, forming a large clustered surface which increased the interaction between the DNA molecules and the NH 2 ligand of γ-APTES.

Label Free DNA Detection Using Large Area Graphene-Based FET Biosensors

ABSTRACTThis work describes the fabrication of highly sensitive graphene-based field effect transistor (FET) biosensors in a cost-effective way and its application in label-free DNA detection. CVD graphene was used to achieve mass production of FET device through photolithography method. Non-covalent functionalization of graphene with 1-Pyrenebutanoic acid succinimidyl ester ensures the high conductivity and sensitivity of the device. The present device could reach the low detection limit as low as 3*10-9 M.

Graphene oxide as an efficient signal-to-background enhancer for DNA detection with a long range resonance energy transfer strategy

A long range resonance energy transfer (LrRET) strategy for label-free and sensitive DNA detection is outlined by introducing graphene oxide (GO) as an efficient signal-to-background enhancer, giving a limit of determination (3σ) of 0.31 nM.