Metal nanoparticles as labels for heterogeneous, chip-based DNA detection

The last decade has witnessed the development of a variety of metal nanoparticle-based techniques for DNA detection. High sensitivity and specificity, miniaturization, and cost-efficient detection are problems addressed by the use of nanoparticle labels in heterogeneous DNA detection schemes. The small label size, established bioconjugation chemistry, and the unusual optical and electrical properties of metal nanoparticles make them unique tools for DNA detection. This paper reviews the different physical characteristics of metal nanoparticles and their implementation in assays. It covers various optical as well as gravimetric, electrochemical and electrical methods for analysing nanoparticle-labelled analytes, and particularly DNA, at sensing surfaces.     

Gold and silica-coated gold nanoparticles as thermographic labels for DNA detection

The infrared emissivity of Au and silica-coated Au nanoparticles (Au NPs) deposited on indium tin oxide substrates was investigated. NPs were irradiated with laser light at a frequency close to the Au plasmon resonance band, and the blackbody radiation emitted as a result was monitored with an IR camera equipped with an InAs array detector. The differences in temperature before and after laser irradiation were recorded (T-jumps) and were found to be directly proportional to the number of particles present on the slide and to the laser power used in the experiment. Coating Au NPs with silica increased the measured T-jumps 2-5 times, depending on the thickness of the silica shell. This was in agreement with the observation that silica has a much higher IR emissivity than Au. Both Au and silica-coated Au NPs were then tested as labels for thermographic DNA detection. Target DNA concentrations as low as 100 pM were recorded when Au NPs were used as labels and as low as 10 pM when silica-coated Au NPs were used.     

On-line determination of the molar ratio between methanol and isobutylene in feedstock of a methyl tertiary butyl ether production plant using near-infrared spectroscopy

The molar ratio between methanol and isobutylene (MRMI) is very important to the operation of methyl tertiary butyl ether (MTBE) production units. A new on-line near-infrared (NIR) analytical system was integrated for monitoring the MRMI in real time and was successfully applied in a rubber plant. Calibration models for methanol and isobutylene were established using partial least squares (PLS). The sample temperature effect on the performance of the models is discussed. The MRMI is calculated by methanol content and isobutylene content predicted by NIR. A large benefit has been obtained by the user through controlling the operation of the unit according to the monitoring of the MRMI of the feedstock in real time.     

Carbon nanoparticles as detection labels in antibody microarrays. Detection of genes encoding virulence factors in Shiga toxin-producing Escherichia coli

The present study demonstrates that carbon nanoparticles (CNPs) can be used as labels in microarrays. CNPs were used in nucleic acid microarray immunoassays (NAMIAs) for the detection of different Shiga toxin-producing Escherichia coli (STEC) virulence factors: four genes specific for STEC (vt1, vt2, eae, and ehxA) and the gene for E. coli 16S (hui). Optimization was performed using a Box-Behnken design, and the limit of detection for each virulence factor was established. Finally, this NAMIA using CNPs was tested with DNA from 48 field strains originating from cattle feces, and its performance was evaluated by comparing results with those achieved by the reference method q-PCR. All factors tested gave sensitivity and specificity values higher than 0.80 and efficiency values higher than 0.92. Kappa coefficients showed an almost perfect agreement (k > 0.8) between NAMIA and the reference method used for vt1, eae, and ehxA, and a perfect agreement (k = 1) for vt2 and hui. The excellent agreement between the developed NAMIA and q-PCR demonstrates that the proposed analytical procedure is indeed fit for purpose, i.e., it is valuable for fast screening of amplified genetic material such as E. coli virulence factors. This also proves the applicability of CNPs in microarrays.     

Study of superparamagnetic nanoparticles as labels in the quantitative lateral flow immunoassay

The superparamagnetic nanoparticles (MNPs) with a series of sizes and magnetite contents are designed and prepared as labels used in lateral flow immunoassay (LFIA) to test if the size and magnetite content of MNPs can affect the performance of LFIA. The result showed that detection time was mainly determined by the size of MNPs, while the signal intensity was closely correlated to the magnetite content of the MNPs. In addition, the magnetic signals intensity remained stable over a long time period. Smaller MNPs with higher magnetite content are first choices as labels to construct a rapid and high-sensitive quantitative LFIA.     

Sensing technique of silver nanoparticles as labels for immunoassay using liquid electrode plasma atomic emission spectrometry

We report the use of liquid electrode plasma-atomic emission spectrometry (LEP-AES) in protein sensing studies employing Ag nanoparticle labeling. LEP-AES requires no plasma gas and no high-power source and is suitable for onsite portable analysis. Human chorionic gonadotropin (hCG) was used as a model target protein, and the immunoreaction in which hCG is sandwiched between two antibodies, one of which is immobilized on the microwell and the second is labeled with Ag nanoparticles, was performed. Sensing occurs at the narrow pass in the center of a quartz chip following oxidative dissolution of the Ag nanoparticles by nitric acid. hCG was analyzed in the range from 10 pg/mL to 1 ng/mL, and the detection limit for hCG was estimated at 1.3 pg/mL (22.8 fM). The proposed detection method has a wide variety of promising applications in metal-nanoparticle-labeled biomolecule detection.     

DNA-wrapped carbon nanotubes as sensitive electrochemical labels in controlled-assembly-mediated signal transduction for the detection of sequence-specific DNA

A novel electrochemical strategy that uses DNA-wrapped carbon nanotubes (CNTs) as electrochemical labels is developed for sensitive and selective detection of sequence-specific DNA. The presence of target DNA mediates the formation of a sandwiched complex between the DNA-wrapped CNT and a hairpin DNA capture probe immobilized on magnetic beads. This allows target-selective collection of the CNT labels by magnetic separation and transfer on the electrode surface modified with an insulating self-assembled monolayer (SAM). After treatment with N,N-dimethylformamide, the collected sandwiched complex releases the bare CNTs and facilitates the removal of magnetic beads from the electrode surface. The bare CNTs can then assemble on the SAM-modified electrode surface and mediate efficient electron transfer between the electrode and the electroactive species in the solution with a strong current signal generated. The results indicate that the developed strategy shows a sensitive response to target DNA with a desirable signal gain and a low detection limit of 0.9 pM. This strategy is also demonstrated to provide excellent differentiation of single-base mismatch in target DNA. It is expected that this electrochemical strategy may hold great potential as a novel platform for clinical diagnostics and genetic analysis.     

Luminescent properties of ZrO2:Tb nanoparticles for applications in neuroscience

In this paper a new generation of non-toxic nanoparticles based on the zirconium oxide doped with 0.5%Tb and co-doped by the range of 0–70% with Y was evaluated for the use as a fluorescent biomarker of neuronal trafficking. The ZrO₂:Tb nanoparticles were created by microwave driven hydrothermal method. Influence of the yttrium content and thermal processing on the Tb³⁺ related luminescence emission was discussed. The higher intensities were achieved, when host was cubic and for the nanoparticles with 33 nm. Presence of yttrium was associated with the energy coupling of the host and dopant, wide excitation band is present at 309 and 322 nm before and after calcination respectively.For the experiment on living primary neurons, nanoparticles doped with 0.5%Tb and 7%Y were chosen based on their luminescence emission intensity. Recently transfer of the nanoparticles through the barriers in the organism including blood–brain barrier following their alimentary absorption was confirmed (Godlewski and Godlewski, 2012). This raised the possibility of the nanoparticle application as a tool in the neuroscience, and the question of potential mechanisms of nanoparticle turnover in neurons. Concentration of 0.001 mg/ml of ZrO₂:0.5%Tb 7%Y in growth medium was added to the primary murine culture medium, and the intracellular trafficking of nanoparticles was observed following 15 min pre-incubation period. ZrO₂:0.5%Tb 7%Y nanoparticles were dynamically absorbed by the neurons and the dynamic passage of transport vesicles containing ZrO₂:0.5%Tb 7%Y nanoparticles was observed along the neuronal processes and in between two neighbouring neurons. Reassuming, the ZrO₂:0.5%Tb 7%Y nanoparticles proved to be biocompatible and a valid tool to assess intracellular trafficking dynamics in the neurobiology.     

Oligonucleotide-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for DNA analysis by hybridization

The highly specific molecular recognition properties of oligonucleotides are combined with the unique optical properties of gold nanoparticles for the development of a dry-reagent strip-type biosensor that enables visual detection of double stranded DNA within minutes. The assay does not require instrumentation and avoids the multiple incubation and washing steps performed in most current assays. Gold nanoparticle reporters with oligo(dT) attached to their surface form an integral part of the strip. Biotinylated PCR products (233 bp or 495 bp) are hybridized (5 min) with a poly(dA)-tailed oligo and applied on the strip, which is then immersed in the appropriate buffer. As the buffer migrates upward, it rehydrates the nanoparticles that are linked to the target DNA through poly(dA)/(dT) hybridization. Capture of the hybrids by immobilized streptavidin in the test zone of the strip generates a characteristic red band. A second red band is formed, by hybridization, in the control zone of the strip to indicate proper test performance. The sensor offers at least 8 times higher detectability than ethidium bromide staining of agarose gels and provides confirmation of the amplified fragments. Quantitative data are obtained by densitometric analysis of the bands. As low as 2 fmol of amplified DNA were detectable by the strip sensor. Also, 500 copies of prostate-specific antigen cDNA were detected by combining PCR and the strip sensor. The sensor was used successfully for detection of hepatitis C virus in plasma samples from 20 patients. The strip detected 16 out of 16 positive samples and gave no signal for 4 samples that were negative for the virus. To our knowledge, this is the first dry-reagent system that makes use of oligonucleotide-conjugated gold nanoparticles as probes.     

Identification and elimination of polysiloxane curing agent interference encountered in the quantification of low-picogram per milliliter methyl tert-butyl ether in blood by solid-phase microextraction headspace analysis

Widespread use of the gasoline additive methyl tert-butyl ether (MTBE) and the subsequent human exposure that follows have led to the need to quantify MTBE in a variety of complex biological matrixes. In this work, we demonstrate our latest MTBE quantification assay for whole blood and uncover previously unidentified contamination sources that prevented routine quantification in the low picogram per milliliter (parts per trillion, ppt) range despite a sensitive and selective analytical approach. The most significant and unexpected sources of contamination were found in reagents and laboratory materials most relevant to sample preparation and quantification. In particular, significant levels of MTBE were identified in sample vial septa that use poly(dimethylsiloxane) (PDMS)-based polymers synthesized with peroxide curing agents having tert-butyl side groups. We propose that MTBE is one of the byproducts of these curing agents, which cross-link PDMS via the methyl side groups. Residual MTBE levels of approximately 20 microg/septa are seen in septa whose formulations use these curing agents. Fortunately, these levels can be significantly reduced (i.e., <0.2 ng/septa) by additional processing. Performance achieved with this sample preparation approach is demonstrated using a mass spectrometry-based method to quantify blood MTBE levels in the low-ppt range.     

Method detection limit determination and application of a convenient headspace analysis method for methyl tert-butyl ether in water

Methyl tert-butyl ether (MTBE) is a common groundwater contaminant, introduced to the environment by leaking petroleum storage tanks, urban runoff, and motorized watercraft. In this study. a simplified (static) headspace analysis method was adapted for determination of MTBE in water samples and soil water extracts. The MDL of the headspace method was calculated to be 2.0 microg L(-1) by the EPA single-concentration design method(1) and 1.2 microg L(-1) by a calibration method developed by Hubaux and Vos (Hubaux, A.; Vos, G. Anal. Chem. 1970,42, 849-855). The MDL calculated with the Hubaux and Vos method was favored because it considers both a true positive and a false positive. The static headspace method was applied to analysis of a tap water sample and a monitoring well sample from a gasoline service station, a river sample, and aqueous extracts from soil excavated during removal of a leaking underground storage tank (LUST). The water samples examined in this study had MTTBE concentrations ranging from 6 to 19 microg L(-1). Aqueous extracts of a soil sample taken from the LUST site had 8 microg L(-1) MTBE.     

Immobilization of Au nanoparticles on Au electrode for hydrazine detection: Using thiolated single-stranded DNA as a linker

In this article, we report a method for effective immobilization of Au nanoparticles (AuNPs) on thiolated single-stranded DNA (thiol-ssDNA) modified Au electrode (AuE) surface via coordination interactions between the nitrogen atoms of DNA bases and AuNPs. It suggests that the resultant AuNP-immobilized AuE exhibits notable catalytic performance for hydrazine oxidation and the loading of AuNPs on the AuE surface and hence the effective catalytic area can be tuned by the immobilization time of thiol-ssDNA and adsorption time of AuNPs. This hydrazine sensor has a fast amperometric response time of less than 4 s. The linear range and detection limit are estimated to be from 0.1 mM to 100 mM (r = 0.998) and 0.56 μM at a signal-to-noise ratio of 3, respectively.     

The use of myristic acid as a ligand of polyethylenimine/DNA nanoparticles for targeted gene therapy of glioblastoma

To establish a gene delivery system for brain targeting, a low molecular weight polyethylenimine (PEI(10?K)) was modified with myristic acid (MC), and complexed with DNA, yielding MC-PEI(10?K)/DNA nanoparticles successfully. The nanoparticles were observed to be successfully taken up by the brains of mice. The transfection efficiency of the nanoparticles was then investigated, and both the in?vitro and in?vivo gene expression of MC-PEI(10?K)/DNA nanoparticles is significantly higher than that of unmodified PEI(10?K)/DNA nanoparticles. The anti-glioblastoma effect of MC-PEI(10?K)/pORF-hTRAIL was demonstrated by the survival time of intracranial U87 glioblastoma-bearing mice. The median survival time of the MC-PEI(10?K)/pORF-hTRAIL group (28 days) was significantly longer than that of the PEI(10?K)/pORF-hTRAIL group (24 days), the MC-PEI(10?K)/pGL(3) group (21 days) and the saline group (22 days). Therefore, our results suggested that MC-PEI(10?K) could be potentially used for brain-targeted gene delivery and in the treatment of glioblastoma.     

Amine-terminated TEG-derived PAMAM dendrimer as template for preparation of gold nanoparticles in water

Nano-sized monodisperse gold particles (AuNPs) have received significant attention in the past decade, due to their unique physical properties and good chemical stability, which can lead to a wide variety of potential applications. In this work, TEG-derived PAMAM dendrimers with amine-terminating groups were synthesized and characterized by ¹H NMR and FT-IR. These dendrimers were investigated as the templates for preparation of gold nanoparticles through the reduction of HAuCl₄ by NaBH₄ in water. Stable gold nanoparticles with diameters around 10 nm were obtained in the presence of G2.0–G5.0 dendrimers and characterized by UV-Vis spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS). The particle size of the produced AuNPs decreased with increasing dendrimer generations. A dendrimer of higher generation has a rigid structure with many end groups on the surface and may play a powerful role in the growth of the AuNPs, as well as having a solid stabilization effect on the AuNPs.     

Amine-terminated TEG-derived PAMAM dendrimer as template for preparation of gold nanoparticles in water

Nano-sized monodisperse gold particles (AuNPs) have received significant attention in the past decade, due to their unique physical properties and good chemical stability, which can lead to a wide variety of potential applications. In this work, TEG-derived PAMAM dendrimers with amine-terminating groups were synthesized and characterized by ¹H NMR and FT-IR. These dendrimers were investigated as the templates for preparation of gold nanoparticles through the reduction of HAuCl₄ by NaBH₄ in water. Stable gold nanoparticles with diameters around 10 nm were obtained in the presence of G2.0 – G5.0 dendrimers and characterized by UV-Vis spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS). The particle size of the produced AuNPs decreased with increasing dendrimer generations. A dendrimer of higher generation has a rigid structure with many end groups on the surface and may play a powerful role in the growth of the AuNPs, as well as having a solid stabilization effect on the AuNPs.     

Biosynthesis of silver nanoparticles using Artemisia annua callus for inhibiting stem‐end bacteria in cut carnation flowers

A biological method for synthesising silver nanoparticles (AgNPs) was developed using the callus extracts from Artemisia annua L. under sunlight at 25,000 lx. The AgNPs were characterised using transmission electron microscopy, atomic force microscope, X‐ray diffraction and Fourier transform infrared spectroscopy. The AgNPs were mostly spherical with the size of 2.1 to 45.2 nm (average 10.9 nm). Pulse treatments of AgNPs at 125, 250 and 500 mg/l for 1 h extended vase life of cut carnation (Dianthus caryophyllus cv. Green Land) flowers. Four dominant bacteria strains Arthrobacter arilaitensis, Kocuria sp., Staphylococcus equorum and Microbacterium oxydans were isolated from the stem‐ends of cut D. caryophyllus flowers. AgNP pulse inhibited significantly bacterial growth in vase solution and cut stem ends during all of the vase period. The bacteria related blockage in the stem‐ends was significantly alleviated by AgNP pulse because of its higher antibacterial efficacy against the dominant bacteria. In addition, ethylene release of cut carnation flowers was inhibited in response to AgNP pulse. This is the first time that the biologically synthesised AgNPs could be applied as a promising preservative agent for cut carnation flowers.Inspec keywords: nanofabrication, silver, nanoparticles, microorganisms, transmission electron microscopy, atomic force microscopy, X‐ray diffraction, Fourier transform infrared spectraOther keywords: biosynthesis, silver nanoparticles, Artemisia annua callus, stem end bacteria, cut carnation flowers, biological method, transmission electron microscopy, atomic force microscope, X‐ray diffraction, Fourier transform infrared spectroscopy, Dianthus caryophyllus cv. Green Land, Arthrobacter arilaitensis, Kocuria sp, Staphylococcus equorum, Microbacterium oxydans, ethylene release, time 1 h, Ag     

Synthesis of calcium peroxide nanoparticles as an innovative reagent for in situ chemical oxidation

Chemical oxidation is one of the many different methods of site remediation that has emerged lately as an alternative method to traditional techniques. According to this research calcium peroxide is suitable choice for contaminant biodegradation in soil and ground water but speed of oxidation reaction between calcium peroxide and contaminant is slow. Synthesis of calcium peroxide in nano size by increased ratio of surface to volume can increase the speed of reaction and solve the problem. We have developed a simple surface modification technique to avoid irreversible agglomeration of calcium peroxide nanoparticles. The technique is based on hydrolysis-precipitation procedure, using CaCl₂ as a precursor. Polyethylene glycol 200 (PEG200) is used as a surface modifier. CaO₂ was identified and studied by characterization techniques, including XRD and TEM. The results indicate the ability of this method for synthesis of new reagent in nano size and improve quality of in situ chemical oxidation. Size determination by TEM image indicates the size of calcium peroxide nanoparticles approximately 15-25 nm.