Real-time multicolor DNA detection with chemoresponsive diffraction gratings and nanoparticle probes

We report a real-time DNA detection method that utilizes single-strand DNA-modified nanoparticle probes and micropatterned chemoresponsive diffraction gratings interrogated simultaneously at multiple laser wavelengths. The surface-bound nanoparticle probe based assay with the chemoresponsive diffraction grating signal transduction scheme results in an experimentally simple DNA detection protocol, displaying attributes of both detection methodologies: the high sensitivity and selectivity afforded by nanoparticle probes and the experimental simplicity, wavelength-dependent resonant enhancement features, and miniaturization potential provided by the diffraction-based sensing technology.     

Mixed DNA-functionalized nanoparticle probes for surface-enhanced Raman scattering-based multiplex DNA detection

Highly stable silver nanoparticle-oligonucleotide conjugates were prepared. Based on the mixed DNA-functionalized silver nanoparticles (AgNPs), multiplex DNA detections were demonstrated successfully by SERS.     

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.     

Optical DNA detection based on gold nanorods aggregation

Sequence-specific DNA detection is important in various biomedical applications such as gene expression profiling, disease diagnosis and treatment, drug discovery and forensic analysis. Herein, the localized surface plasmon resonance properties of unmodified gold nanorods (GNRs) in 1 mM cetyltrimethyl-ammonium bromide solution were used for sensing DNA sequences, with good simplicity and sensitivity. The intensity of typical plasmon resonance absorption bands of the GNRs decreased with increasing cDNA concentration. The detection of a 30-mer single-stranded oligonucleotide as a model reached a detection limit of about 0.1 pM. This study will be significant for as-prepared GNRs for future application in biological systems.     

Self-assembled nanoparticle probes for recognition and detection of biomolecules

Colloidal gold nanocrystals have been used to develop a new class of nanobiosensors that is able to recognize and detect specific DNA sequences and single-base mutations in a homogeneous format. At the core of this biosensor is a 2.5-nm gold nanoparticle that functions as both a nano-scaffold and a nano-quencher (efficient energy acceptor). Attached to this core are oligonucleotide molecules labeled with a thiol group at one end and a fluorophore at the other. This hybrid bio/inorganic construct is found to spontaneously assemble into a constrained arch-like conformation on the particle surface. Binding of target molecules results in a conformational change, which restores the fluorescence of the quenched fluorophore. Unlike conventional molecular beacons with a stem-and-loop structure, the nanoparticle probes do not require a stem, and their background fluorescence increases little with temperature. In comparison with the organic quencher Dabcyl (4,4'-dimethylaminophenyl azo benzoic acid), metal nanoparticles have unique structural and optical properties for new applications in biosensing and molecular engineering.     

Gold nanoparticle based FRET assay for the detection of DNA cleavage

We report gold nanoparticle based FRET assay to monitor the cleavage of DNA by nucleases. Fluorescence signal enhancement is observed by a factor of 120 after the cleavage reaction in the presence of S1 nuclease. The mechanism of distant dependent fluorescence quenching has been discussed. Our experimental results on distance dependent fluorescence quenching match quite well with theoretical findings obtained from the fluorescence quenching model by Gersten and Nitzan (Surf. Sci. 1985, 158, 165). Our experimental observation paradigm for the design of optical based molecular ruler strategies at distances more than double the distances achievable using traditional dipole-dipole Columbic energy transfer based methods.     

Electrochemical stripping detection of DNA hybridization based on cadmium sulfide nanoparticle tags

We report on the detection of DNA hybridization in connection to cadmium sulfide nanoparticle tracers and electrochemical stripping measurements of the cadmium. A nanoparticle-promoted cadmium precipitation is used to enlarge the nanoparticle tag and amplify the stripping DNA hybridization signal. In addition to measurements of the dissolved cadmium ion we demonstrate solid-state measurements following a ‘magnetic’ collection of the magnetic-bead/DNA-hybrid/CdS-tracer assembly onto a thick-film electrode transducer. The new protocol combines the amplification features of nanoparticle/polynucleotides assemblies and highly sensitive stripping potentiometric detection of cadmium, with an effective magnetic isolation of the duplex. The low detection limit (100 fmol) is coupled to good reproducibility (RSD=6%). Prospects for using binary inorganic colloids for multi-target detection are discussed.     

Colorimetric detection of DNA at the nanomolar level based on enzyme-induced gold nanoparticle de-aggregation

The authors describe a colorimetric method for the determination of DNA based on the deaggregation of gold nanoparticles (AuNPs) induced by exonuclease III (Exo III). DNA amplification is accomplished by Exo III to generate large quantities of the residual DNA. Residual DNA tethers onto the surfaces of AuNPs which prevents their aggregation. Hence, the color of the solution is red. However, in the absence of DNA, salt-induced aggregation is not prevented, and the bluish-purple color of the aggregated AuNPs is observed. The ratio of absorbances at 525 and 625 nm increases up to 150 nM DNA concentrations, and the LOD is as low as 3.0 nM. It is shown that the presence of 300 nM concentrations of random DNA (with a mass up to 10-fold that of target DNA) does not interfere. The method was successfully applied to the analysis of DNA in spiked serum samples. The method is simple, reliable, and does not require complicated amplification steps and expensive instrumentation.

Open image in new window

Graphical abstract Schematic of a sensing strategy for DNA detection by exonuclease III-induced deaggregation of gold nanoparticles. DNA concentrations as  low as 3 nM can be detected via colorimetric monitoring of the color change from red to purple-blue.

     

An electrochemical DNA hybridization detection assay based on a silver nanoparticle label

A novel, sensitive electrochemical DNA hybridization detection assay, using silver nanoparticles as the oligonucleotide labeling tag, is described. The assay relies on the hybridization of the target DNA with the silver nanoparticle-oligonucleotide DNA probe, followed by the release of the silver metal atoms anchored on the hybrids by oxidative metal dissolution and the indirect determination of the solubilized Ag(I) ions by anodic stripping voltammetry (ASV) at a carbon fiber ultramicroelectrode. The influence of the relevant experimental variables, including the surface coverage of the target oligonucleotide, the duration of the silver dissolution steps and the parameters of the electrochemical stripping measurement of the silver(I) ions, is examined and optimized. The combination of the remarkable sensitivity of the stripping metal analysis at the microelectrode with the large number of silver(I) ions released from each DNA hybrid allows detection at levels as low as 0.5 pmol L(-1) of the target oligonucleotides.     

A thermodynamic investigation into the binding properties of DNA functionalized gold nanoparticle probes and molecular fluorophore probes

We report the first quantitative analysis of the oligonucleotide binding thermodynamics for DNA functionalized gold nanoparticle probes and compare our findings to molecular fluorophore probes on a sequence-for-sequence basis. With proper design, nanoparticle probes show significantly increased binding over molecular fluorophore probes under identical conditions. This is significant because probe binding strength directly influences detection sensitivity limits.     

Structural study on gold nanoparticle functionalized with DNA and its non-cross-linking aggregation

Hybridization of DNA tethered on colloidal nanoparticles with fully matched complementary one induces the aggregation of the particles in a non-cross-linking configuration. Here, we performed a structural study on DNA-functionalized gold nanoparticle and its non-cross-linking aggregation mainly using synchrotron radiation small-angle X-ray scattering. To understand the non-cross-linking aggregation, the nanoparticles with various DNA lengths and core sizes were used. In the aggregation, the surface distance between the gold nanoparticles increased with the length of DNA duplex, although the increment of the distance per base pair was not constant and showed the tendency to become small with increasing DNA length, meaning the interdigitation of DNA layers. The aggregation was also found to occur between the identical cores, without being affected by tethered DNA. Furthermore, it was proved that the relative increase in DNA length to core size leads to the increase in colloidal stability. Even the nanoparticles with full-matched DNA duplex were dispersed stably. These facts suggested that van der Waals interaction between core particles rather than end-to-end stacking between DNA duplexes is a dominant attractive interaction. The steric repulsion force arising from entropic loss of thermal fluctuation of DNA molecules might be a key factor to characterize the non-cross-linking aggregation.     

Multiplexed detection of protein cancer markers with biobarcoded nanoparticle probes

We have developed the chemistry for preparing a universal probe and the appropriate nano- and microparticle labels that can be used to do highly selective multiplexed detection of three protein cancer markers at low-femtomolar concentration in buffer and serum media. The approach relies on a new multiplexed version of the biobarcode amplification method and offers new opportunities for studying multiple protein markers in a single sample. This could lead to new forms of disease diagnosis and monitoring disease recurrence in a variety of settings.     

Gold nanoparticle probes for the detection of mercury, lead and copper ions

Monitoring the levels of potentially toxic metal (PTM) ions (e.g., Hg(2+), Pb(2+), Cu(2+)) in aquatic ecosystems is important because these ions can have severe effects on human health and the environment. Gold (Au) nanomaterials are attractive sensing materials because of their unique size- and shape-dependent optical properties. This review focuses on optical assays for Hg(2+), Pb(2+), and Cu(2+) ions using functionalized Au nanomaterials. The syntheses of functionalized Au nanomaterials are discussed. We briefly review sensing approaches based on changes in absorbance resulting from metal ion-induced aggregation of Au nanoparticles (NPs) or direct deposition of metal ions onto Au NPs. The super-quenching properties of Au NPs allow them to be employed in 'turn on' and 'turn off' fluorescence approaches for the sensitive and selective detection of Hg(2+), Pb(2+), and Cu(2+) ions. We highlight approaches based on fluorescence quenching through analyte-induced aggregation or the formation of metallophilic complexes of Au nanodots (NDs). We discuss the roles of several factors affecting the selectivity and sensitivity of the nanosensors toward the analytes: the size of the Au nanomaterial, the length and sequence of the DNA or the nature of the thiol, the surface density of the recognition ligand, and the ionic strength and pH of the buffer solution. In addition, we emphasize the potential of using new nanomaterials (e.g., fluorescent silver nanoclusters) for the detection of PTM ions.     

Enhanced electrochemiluminescence employed for the selective detection of methyl parathion based on a zirconia nanoparticle film modified electrode

A simple, rapid and sensitive electrochemiluminescence (ECL) sensor was proposed for direct measurements of methyl parathion (MP) based on the strong affinity of a nano zirconia particles (ZrO(2) NPs) modified film on the electrode to the phosphoric group. ZrO(2) NPs, which could provide a larger absorption area to immobilize organophosphorus, was firstly modified on the glassy carbon electrode surface to prepare the proposed ECL sensor (ZrO(2)/GC). Subsequently, the ZrO(2)/GC electrode was scanned from -0.8 to +0.6 V to obtain the background signal at 0.44 V in a luminol/KCl solution. Then, a certain concentration of MP was added to an aqueous solution for 240 s, which was absorbed onto the ZrO(2)/GC electrode surface. Moreover, the MP absorbed on the surface of the ZrO(2)/GC electrode enhanced the ECL signal of luminol in the luminol/KCl solution, which increased with the concentration of MP. As a result, a novel ECL sensor was obtained in a luminol/KCl solution. The MP was determined in the range of from 3.8 × 10(-11) to 3.8 × 10(-6) mol L(-1), with a low detection limit of 1.27 × 10(-11) mol L(-1) (S/N = 3). The proposed ECL sensor performance for MP detection will open a new field in the application of rapid and screen detection of ultra-trace amounts of organ phosphorus pesticides (OPs) of vegetables used in farm markets.     

Biomolecule detection via target mediated nanoparticle aggregation and dielectrophoretic impedance measurement

A new biosensing system is described that is based on the aggregation of nanoparticles by a target biological molecule and dielectrophoretic impedance measurement of these aggregates. The aggregation process was verified within a microchannel via fluorescence microscopy, demonstrating that this process can be used in a real time sensor application. Positive dielectrophoresis is employed to capture the nanoparticle aggregates at the edge of thin film electrodes, where their presence is detected either by optical imaging via fluorescence microscopy or by measuring the change in electrical impedance between adjacent electrodes. The electrical detection mechanism demonstrates the potential for this method as a micro total analysis system (microTAS).     

DNA sensor: A novel electrochemical gene detection method using carbon electrode immobilized DNA probes

Abstract

The authors have developed a novel, rapid, convenient, and specific gene detection method, named the ‘DNA sensor,’ using a graphite electrode loaded with DNA probes. Synthesized oligonucleotide (5-TGCAGTTCCGGTGGCTGATC-3′) complementary to oncogene v-myc was employed for a model probe. The oligonucleotide was chemically adsorbed on a basal plane pyrolytic graphite (BPPG) electrode. The sensor was able to be applied to a hybridization reaction (40°C) in a linearized pVM623 solution carrying the Pst I fragment of v-myc (1.5 kbp).

After the hybridization reaction, the sensor was immersed into an acridine orange solution (1 μM) and washed with a phosphate buffer (pH 7.0). Acridine orange intercalated between base pairs of the formed double stranded DNAs on the electrode. The anodic peak potential of acridine orange that interacted with the DNAs on the electrode was measured. The positive shift of the peak potential increased in proportional to the pVM623 concentration in the hybridization reaction. 10^?1 g/ml of pVM623 was able to be detected in the buffer solution using the sensor. This gene detection was completed within an hour.     

Sensitive and selective detection of glutathione based on resonance light scattering using sensitive gold nanoparticles as colorimetric probes

In this paper, we reported the development of a highly sensitive and selective resonance light scattering (RLS) technique for glutathione using gold nanoparticle probes. The assay relies upon the distance-dependent optical properties of gold nanoparticles, the self-assembly of glutathione on gold nanoparticles, and the interaction of a 2 : 1 glutathione-Cu(2+) complex. In the presence of Cu(2+), glutathione could rapidly induce the aggregation of gold nanoparticles, thereby resulting in greatly enhanced RLS intensity and red-to-blue (or purple) color change. The concentration of glutathione can be determined by the naked eye or a fluorescence spectrometer. Under the optical conditions, the detection of glutathione can be finished within 20 min, and the detection limit of 10 nM can be reached. The concentration range of the probe is 40-280 nM. The proposed method holds a specific selectivity toward glutathione and it is applied to the detection of glutathione in human serum with satisfactory results. In addition, the assay shows great potential application for disease-associated biomarkers, and it will meet the great demand for amino acid determination in fields such as food processing, biochemistry, pharmaceutical, and clinical analysis.