A real-time PCR-based method for determining the surface coverage of thiol-capped oligonucleotides bound onto gold nanoparticles

Here we report a real-time PCR-based method for determining the surface coverage of dithiol-capped oligonucleotides bound onto gold nanoparticles alone and in tandem with antibody. The detection of gold nanoparticle-bound DNA is accomplished by targeting the oligonucleotide with primer and probe binding sites, amplification of the oligonucleotide by PCR, and real-time measurement of the fluorescence emitted during the reaction. This method offers a wide dynamic range and is not dependant on the dissociation of the oligonucleotide strands from the gold nanoparticle surface; the fluorophore is not highly quenched by the gold nanoparticles in solution during fluorescence measurements. We show that this method and a fluorescence-based method give equivalent results for determining the surface coverage of oligonucleotides bound onto 13 or 30 nm gold nanoparticles alone and in tandem with antibody. Quantifying the surface coverage of immobilized oligonucleotides on metallic nanoparticle surfaces is important for optimizing the sensitivity of gold nanoparticle-based detection methods and for better understanding the interactions between thiol-functionalized oligonucleotides and gold nanoparticles.     

Efficient manipulation of nanoparticle-bound DNA via restriction endonuclease

As a programmable biopolymer, DNA has shown great potential in the fabrication and construction of nanometer-scale assemblies and devices. In this report, we described a strategy for efficient manipulation of gold nanoparticle-bound DNA using restriction endonuclease. The digestion efficiency of this restriction enzyme was studied by varying the surface coverage of stabilizer, the size of nanoparticles, as well as the distance between the nanoparticle surface and the enzyme-cutting site of particle-bound DNA. We found that the surface coverage of stabilizer is crucial for achieving high digestion efficiency. In addition, this stabilizer surface coverage can be tailored by varying the ion strength of the system. Based on the results of polyacrylamide gel electrophoresis and fluorescent study, a high digestion efficiency of 90+% for particle-bound DNA was achieved for the first time. This restriction enzyme manipulation can be considered as an additional level of control of the particle-bound DNA and is expected to be applied to manipulate more complicated nanostructures assembled by DNA.     

Grating-based surface plasmon resonance detection of core-shell nanoparticle mediated DNA hybridization

In this report, we have investigated enhanced surface plasmon resonance (SPR) detection of DNA hybridization using gold core - silica shell nanoparticles in localized plasmonic fields. The plasmonic fields were localized by periodic linear gratings. Experimental results measured for hybridization of 24-mer single-stranded DNA oligomers suggest that core-shell nanoparticles (CSNPs) on gratings of 400 nm period provide enhanced optical signatures by 36 times over conventional thin film-based SPR detection. CSNP-mediated DNA hybridization produced 3 times larger angular shift compared to gold nanoparticles of the same core size. We have also analyzed the effect of structural variation. The enhancement using CSNPs was associated with increased surface area and index contrast that is combined by improved plasmon coupling with localized fields on gratings. The combined approach for conjugated measurement of a biomolecular interaction on grating structures is expected to lower the limit of detection to the order of a few tens of fg/mm(2).     

Polymerase chain reaction of nanoparticle-bound primers

Using one or two primers respectively bound to the surface of Au nanoparticles (AuNPs) or magnetic nanoparticles (MNPs), polymerase chain reaction (PCR) based on nanoparticles was systemically studied, agarose gel electrophoresis and atomic force microscopy (AFM) were respectively used to detect and observe the PCR product. The results obtained indicated that with either one or two primers respectively bound to the nanoparticle surface, PCR can proceed successfully under optimized condition and is subject to certain rules, consequently a symmetric PCR technique and an asymmetric PCR technique based on nanoparticles have been developed. A kind of nanostructured aggregates can be constructed by a symmetric PCR using two nanoparticle-bound primers.     

Elution of labile fluorescent dye from nanoparticles during biological use

Cells act as extremely efficient filters for elution of unbound fluorescent tags or impurities associated with nanoparticles, including those that cannot be removed by extensive cleaning. This has consequences for quantification of nanoparticle uptake and sub-cellular localization in vitro and in vivo as a result of the presence of significant amount of labile dye even following extensive cleaning by dialysis. Polyacrylamide gel electrophoresis (PAGE) can be used to monitor the elution of unbound fluorescent probes from nanoparticles, either commercially available or synthesized in-house, and to ensure their complete purification for biological studies, including cellular uptake and sub-cellular localisation. Very different fluorescence distribution within cells is observed after short dialysis times versus following extensive dialysis against a solvent in which the free dye is more soluble, due to the contribution from free dye. In the absence of an understanding of the presence of residual free dye in (most) labeled nanoparticle solutions, the total fluorescence intensity in cells following exposure to nanoparticle solutions could be mis-ascribed to the presence of nanoparticles through the cell, rather than correctly assigned to either a combination of free-dye and nanoparticle-bound dye, or even entirely to free dye depending on the exposure conditions (i.e. aggregation of the particles etc). Where all of the dye is nanoparticle-bound, the particles are highly localized in sub-cellular organelles, likely lysosomes, whereas in a system containing significant amounts of free dye, the fluorescence is distributed through the cell due to the free diffusion of the molecule dye across all cellular barriers and into the cytoplasm.     

Assays Based on Differential Adsorption of Single-stranded and Double-stranded DNA on Unfunctionalized Gold Nanoparticles in a Colloidal Suspension

Under certain conditions, single-stranded DNA adsorbs to negatively charged gold nanoparticles in a colloid whereas double-stranded DNA does not. We present evidence that this phenomenon can be explained by the difference in their electrostatic properties that in turn reflects conformational differences. The ability to discriminate the hybridization state of DNA on the basis of adsorption behavior can be utilized to design simple colorimetric and fluorimetric assays that take advantage of plasmon resonance in the gold nanoparticles. We present examples where we detect specific target sequences in oligonucleotides and in genomic DNA. Because conformational changes in special DNA sequences can also be induced by analytes such as potassium, we report a potassium ion detection scheme based on the same principle.     

Deoxyribonucleic acid hybridization studies of Exophiala dermatitidis and Exophiala jeanselmei

Exophiala dermatitidis and Exophiala jeanselmei share similar morphological features and have been confused with each other. To clarify the relationship between the two fungi, we conducted a deoxyribonucleic acid (DNA)-DNA hybridization study using a dot blot method. Between E. dermatitidis and E. jeanselmei, only a very low level of DNA relatedness was seen and it was confirmed that these two fungi are distinct species based on DNA similarity. Close correspondence of DNA from the isolates of E. dermatitidis was obtained, whereas the isolates of E. jeanselmei were divided into 6 groups according to their DNA similarity and a possibility was shown that E. jeanselmei is composed of genetically heterogeneous groups. The subdivision of the species E. jeanselmei by the DNA-DNA hybridization method was in agreement with serotyping exoantigens. This result suggests that DNA-DNA hybridization studies provide an excellent tool for the identification and grouping of pathogenic dematiaceous fungi.     

Characterization of DNA chips by nanogold staining

DNA microarray is an important tool in biomedical research. Up to now, there are no chips that can allow both quality analysis and hybridization using the same chip. It is risky to draw conclusions from results of different chips if there is no knowledge of the quality of the chips before hybridization. In this article, we report a colorimetric method to do quality control on an array. The quality analysis of probe spots can be obtained by using gold nanoparticles with positive charges to label DNA through electrostatic attraction. The probe spots can also be detected by a simple personal computer scanner. Gold nanoparticles deposited on a glass surface can be dissolved in bromine-bromide solution. The same microarray treated with gold particles staining and destaining can still be used for hybridization with nearly the same efficiency. This approach makes quality control of a microarray chip feasible and should be a valuable tool for biomarker discovery in the future.     

SPR-based DNA Detection with Metal Nanoparticles

In this short review paper, we summarize some of our ideas to utilize gold nanoparticles for the enhancement of surface plasmon resonance signals on DNA microarray. The hybridization of target-DNA capped gold nanoparticles with probe DNA on surface provides ca. ten times stronger optical contrast compared with that of target-DNA molecules. Our simulation result based on the Maxwell-Garnet theory explains well our experimental data and proves a potential of metallic nanoparticles for the substantial sensitivity enhancements for biosensor application in DNA diagnostics and bio-affinity studies, which leads to the fabrication of high resolution DNA microarrays.     

Inhibition of DNA hybridization by small metal nanoparticles

The inhibition of DNA hybridization by small metal nanoparticles has been examined in detail. DNA melting point analysis showed that the oligonucleotides adsorb strongly and nonspecifically on small metal nanoparticles, inhibiting the hybridization of complementary DNA sequences in common buffered solutions. The nonspecific interaction is even strong enough to disrupt pre-existing hydrogen bonds in short double-stranded DNA. The nonspecific interaction could be weakened by increasing the particle size. As an example, a core-shell assisted method was used to successfully assemble Pt nanoparticles by DNA hybridization that could not be done otherwise.     

Optical detection of DNA hybridization based on fluorescence quenching of tagged oligonucleotide probes by gold nanoparticles

A novel system for the detection of DNA hybridization in a homogeneous format is developed. This method is based on fluorescence quenching by gold nanoparticles used as both nanoscaffolds for the immobilization of capture sequences and nanoquenchers of fluorophores attached to detection sequences. The oligonucleotide-functionalized gold nanoparticles are synthesized by derivatizing the colloidal gold solution with 5'-thiolated 12-base oligonucleotides. Introduction of sequence-specific target DNAs (24 bases) into the mixture containing dye-tagged detection sequences and oligonucleotide-functionalized gold nanoparticles results in the quenching of carboxytetramethylrhodamine-labeled DNA fluorescence because DNA hybridization occurs and brings fluorophores into close proximity with oligonucleotide-functionalized gold nanoparticles. The quenching efficiency of fluorescence increases with the target DNA concentration and provides a quantitative measurement of sequence-specific DNA in sample. A linearity is obtained within the range from 1.4 to 92 nM. The target sequence is detected down to 2 nM. This new system not only overcomes many of the drawbacks inherent in radioisotopic measurement or enzyme-linked assay but also avoids the requirement for the stem-loop structure compared with conventional molecular beacons. Furthermore, the background signal that is defined as fluorescence quenching arising from electrostatic attraction between positively charged fluorophores and negatively charged gold nanoparticles is comparatively low due to electrostatic repulsion between negatively charged oligonucleotides. In addition, this is a homogeneous assay that can offer the potential to be monitored in real time, be amenable to automation, eliminate washing steps, and reduce the risk of contamination.     

Electrochemical genosensors for biomedical applications based on gold nanoparticles

Two gold nanoparticles-based genomagnetic sensors designs for detection of DNA hybridization are described. Both assays are based on a magnetically induced direct electrochemical detection of gold tags on magnetic graphite-epoxy composite electrodes. The first design is a two strands assay format that consists of the hybridization between a capture DNA strand which is linked with paramagnetic beads and another DNA strand related to BRCA1 breast cancer gene used as a target which is coupled with streptavidin-gold nanoparticles. The second genomagnetic sensor design is a sandwich assay format with more application possibilities. A cystic fibrosis related DNA strand is used as a target and sandwiched between two complementary DNA probes: the first one linked with paramagnetic beads and a second one modified with gold nanoparticles via biotin-streptavidin complexation reactions. The electrochemical detection of gold nanoparticles by differential pulse voltammetry was performed in both cases. The developed genomagnetic sensors provide a reliable discrimination against noncomplementary DNA as well against one and three-base mismatches. Optimization parameters affecting the hybridization and analytical performance of the developed genosensors are shown for genomagnetic assays of DNA sequences related with the breast cancer and cystic fibrosis genes.     

Identification and grouping of bacteria by numerical analysis of their electrophoretic protein patterns.

Improved methods for the identification and grouping of bacteria by polyacrylamide gel electrophoresis of soluble proteins are described. Electrophoretic protein patterns were obtained in rigorously standardized comditions. The results were much more reproducible than any described previously. Some of the factors affecting reproducibility were; growth conditions, time and speed of centrifugation of extracts, and conditions of gel electrophoresis. Protein patterns were compared by computing correlation coefficients from normalized densitometric tracings and clustering the strains by the unweighted average pair group method. As model systems, both Agrobacterium and Zymomonas were used because of differences in the sharpness of the peaks. The methodwas applied to 42 Agrobacterium strains. The agreement with the results of clustering by either phenotypic tests or DNA:DNA hybridization was excellent. Computerized comparisons of electrophoretic protein patterns can be a fast, easy and powerful tool for classification and identification of bacteria.     

Surface Plasmon Resonance Studies of the Hybridization Behavior of DNA-Modified Gold Nanoparticles with Surface-Attached DNA Probes

Colloidal gold nanoparticles (AuNPs) have been extensively investigated as amplification tags to improve the sensitivity of surface plasmon resonance (SPR) biosensors. When using the so-called AuNP-enhanced SPR technique for DNA detection, the density of single-stranded DNA (ssDNA) on both the AuNPs and planar gold substrates is of crucial importance. Thus, in this work, we carried out a systematical study about the influence of surface ssDNA density onto the hybridization behavior of various DNA-modified AuNPs (DNA-AuNPs) with surface-attached DNA probes by using surface plasmon resonance spectroscopy. The lateral densities of the ssDNA on both the AuNPs and planar gold substrates were controlled by using different lengths of oligo-adenine sequence (OAS) as anchoring group. Besides SPR measurements, the amount of the captured DNA-AuNPs after the hybridization was further identified via atomic force microscope (AFM). SPR and AFM results clearly indicated that a higher ssDNA density on either the AuNPs or the gold substrates would give rise to better hybridization efficiency. Moreover, SPR data showed that the captured DNA-AuNPs could not be removed from SPR sensor surfaces using various dehybridization solutions regardless of surface ssDNA density. Consequently, it is apparent that the hybridization behavior of DNA-AuNPs was different from that of solution-phase ssDNA. Based on these data, we hypothesized that both multiple recognitions and limited accessibility might account for the hybridization of DNA-AuNPs with surface-attached ssDNA probes.

     

Optimization of "Photosens" pharmakokinetics by biodegradable nanospheres]

The present study is dedicated to investigation of pharmacokinetics of the colloidal delivery system based on polybutylcyanoacrylate nanoparticles for the II generation photosensitizer Photosense. Free or nanoparticle-bound Photosense was injected intravenously in healthy rats in the dose 15 mg/kg. It was shown that pharmacokinetic curve of the free drug was characterized by peak concentration while plasma concentrations of nanoparticulate Photosense were relatively steady. Elimination of nanoparticulate Photosense was more rapid comparing to the free drug. It is noteworthy that nanoparticles did not enhance liver uptake of the drug. Lung level of nanoparticulate drug was found to be lower and spleen uptake was enhanced. More important is the fact that nanoparticles provided two-fold decrease of Photosense skin concentration which is potentially important for decrease of drug-related skin phototoxicity. The above data provide evidence that optimization of Photosense pharmacokinetic parameters could be achieved by the use of nanoparticles.     

Sex determination based on amelogenin DNA by modified electrode with gold nanoparticle

We have developed a simple and renewable electrochemical biosensor based on carbon paste electrode (CPE) for the detection of DNA synthesis and hybridization. CPE was modified with gold nanoparticles (AuNPs), which are helpful for immobilization of thiolated bioreceptors. AuNPs were characterized by scanning electron microscopy (SEM). Self-assembled monolayers (SAMs) of thiolated single-stranded DNA (SH–ssDNA) of the amelogenin gene was formed on CPE. The immobilization of the probe and its hybridization with the target DNA was optimized using different experimental conditions. The modified electrode was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The electrochemical response of ssDNA hybridization and DNA synthesis was measured using differential pulse voltammetry (DPV) with methylene blue (MB) as an electroactive indicator. The new biosensor can distinguish between complementary and non-complementary strands of amelogenin ssDNA. Genomic DNA was extracted from blood and was detected based on changes in the MB reduction signal. These results demonstrated that the new biosensor could be used for sex determination. The proposed biosensor in this study could be used for detection and discrimination of polymerase chain reaction (PCR) products of amelogenin DNA.     

Independently-addressable micron-sized biosensor elements

With the continuing development of micro-total analysis systems and sensitive biosensing technologies, it is often desirable to immobilize biomolecules onto a surface in a small well-defined area. A novel method was developed to electrochemically attach DNA probes to micron-sized regions of a gold surface using biotin-LC-hydrazide (BH). Previously, we have found that the radical produced during the oxidation of BH will attach to a wide variety of electroactive surfaces. An array of micron-sized gold band electrodes (75 microm wide) was fabricated onto glass microscope slides and BH was deposited onto each electrode through the application of an oxidizing potential. Subsequent attachment of avidin to the biotinylated surface created the 'molecular sandwich' architecture necessary for further immobilization of biotinylated biomolecules to the surface. In this work, we utilized biotinylated DNA probes of varying sequence to illustrate the specificity of the attachment scheme. The immobilization of avidin, DNA probe, and hybridization of DNA target is visualized with fluorescence tags and the spatially selective attachment and hybridization of unique DNA sequences is demonstrated.     

Detecting minimal traces of DNA using DNA covalently attached to superparamagnetic nanoparticles and direct PCR-ELISA

A single bond covalent immobilization of aminated DNA probes on magnetic particles suitable for selective molecular hybridization of traces of DNA samples has been developed. Commercial superparamagnetic nanoparticles containing amino groups were activated by coating with a hetero-functional polymer (aldehyde-aspartic-dextran). This new immobilization procedure provides many practical advantages: (a) DNA probes are immobilized far from the support surface preventing steric hindrances; (b) the surface of the nanoparticles cannot adsorb DNA ionically; (c) DNA probes are bound via a very strong covalent bond (a secondary amine) providing very stable immobilized probes (at 100 degrees C, or in 70% formamide, or 0.1N NaOH). Due to the extreme sensitivity of this purification procedure based on DNA hybridization, the detection of hybridized products could be coupled to a PCR-ELISA direct amplification of the DNA bond to the magnetic nanoparticles. As a model system, an aminated DNA probe specific for detecting Hepatitis C Virus cDNA was immobilized according to the optimised procedure described herein. Superparamagnetic nanoparticles containing the immobilized HCV probe were able to give a positive result after PCR-ELISA detection when hybridized with 1 mL of solution containing 10(-18) g/mL of HCV cDNA (two molecules of HCV cDNA). In addition, the detection of HCV cDNA was not impaired by the addition to the sample solution of 2.5 million-fold excess of non-complementary DNA. The experimental data supports the use of magnetic nanoparticles containing DNA probes immobilized by the procedure here described as a convenient and extremely sensitive procedure for purification/detection DNA/RNA from biological samples. The concentration/purification potential of the magnetic nanoparticles, its stability under a wide range of conditions, coupled to the possibility of using the particles directly in amplification by PCR greatly reinforces this methodology as a molecular diagnostic tool.     

Direct DNA conjugation to star polymers for controlled reversible assemblies

Polymer biomolecule hybrids represent a powerful class of highly customizable nanomaterials. Here, we report star-polymer conjugates with DNA using a "ligandless" Cu(I) promoted azide-alkyne cycloaddition click reaction. The multivalency of the star-polymer architecture allows for the concomitant conjugation of other molecules along with the DNA, and the conjugation method provides control over the DNA orientation. The star-polymer DNA nanoparticles are shown to assemble into higher-order nanoassemblies through hybridization. Further, we show that the DNA strands can be utilized in controlled disassembly of the nanostructures.     

Green-synthesized gold nanoparticles decorated graphene sheets for label-free electrochemical impedance DNA hybridization biosensing

Sensitive electrochemical impedance assay of DNA hybridization by using a novel graphene sheets platform was achieved. The graphene sheets were firstly functionalized with 3,4,9,10-perylene tetracarboxylic acid (PTCA). PTCA molecules separated graphene sheets efficiently and introduced more negatively-charged -COOH sites, both of which were beneficial to the decoration of graphene with gold nanoparticles. Then amine-terminated ionic liquid (NH?-IL) was applied to the reduction of HAuCl? to gold nanoparticles. The green-synthesized gold nanoparticles, with the mean diameter of 3 nm, dispersed uniformly on graphene sheets and its outer layer was positively charged imidazole termini. Due to the presence of large graphene sheets and NH?-IL protected gold nanoparticles, DNA probes could be immobilized via electrostatic interaction and adsorption effect. Electrochemical impedance value increased after DNA probes immobilization and hybridization, which was adopted as the signal for label-free DNA hybridization detection. Unlike previously anchoring DNA to gold nanoparticles, this label-free method was simple and noninvasive. The conserved sequence of the pol gene of human immunodeficiency virus 1 was satisfactorily detected via this strategy.