Amplified chemiluminescence surface detection of DNA and telomerase activity using catalytic nucleic acid labels

A G-rich nucleic acid sequence binds hemin and yields a biocatalytic complex (DNAzyme) of peroxidase activity, namely, the biocatalyzed generation of chemiluminescence in the presence of H(2)O(2) and luminol. The DNAzyme is used as a label for the amplified detection of DNA, or for the analysis of telomerase activity in cancer cells, using chemiluminescence as an output signal. In one configuration, the analyzed DNA is hybridized with a primer nucleic acid that is associated with a Au surface, and the DNAzyme label is hybridized with the surface-confined analyte DNA. The DNA is analyzed with a detection limit of approximately 1 x 10(-)(9) M. In the second system, telomerase from HeLa cancer cells induces telomerization of a primer associated with a Au surface and the complementary DNAzyme units are hybridized with the telomere to yield the chemiluminescence. The detection limit of the system corresponds to 1000 HeLa cells in the analyzed sample.     

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.     

Nucleic acid-functionalized Pt nanoparticles: Catalytic labels for the amplified electrochemical detection of biomolecules

Nucleic acid-functionalized Pt nanoparticles (Pt-NPs) act as catalytic labels for the amplified electrochemical detection of DNA hybridization and aptamer/protein recognition. Hybridization of the nucleic acid-modified Pt-NPs with a sensing nucleic acid/analyte DNA complex associated with an electrode enables the amperometric, amplified, detection of the DNA by the Pt NP electrocatalyzed reduction of H2O2 (sensitivity limit, 1 x 10(-11) M). Similarly, the association of aptamer-functionalized Pt- NPs to a thrombin aptamer/thrombin complex associated with an electrode allowed the amplified, electrocatalytic detection of thrombin with a sensitivity limit corresponding to 1 x 10(-9) M.     

Preparation of SiO2/(PMMA/Fe3O4) nanoparticles using linolenic acid as crosslink agent for nucleic acid detection using chemiluminescent method

Usually, magnetic nanoparticles (MNPs) are prepared based on the famous St?ber process in which divinylbenzene (DVA) is often used as a crosslink agent to synthesize SiO2/(PMMA/Fe3O4) nanoparticles. Compared with DVA, linolenic acid (LNA) is innoxious and can polymerize more easily for it has three unsaturated double bonds. In this paper, LNA was used as a new crosslink agent instead of DVA to synthesize the SiO2/(PMMA/Fe3O4) nanoparticles. The results showed that the core-shell structure could be observed obviously. The sizes of nanoparticles with core-shell structure range from 200 to 500 nm. The DNA probes which was immobilized on the surface of MNPs were used to capture the biotin modified complementary sequence of the probe, and the formed complexes were bonded with streptavidin-modified alkaline phosphatase (SA-AP). Finally the chemiluminescent signals were detected by adding 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1, 2-dioxetane (AMPPD) which was the substrate reagent of AP. The specificity and sensitivity of this approach were investigated in this paper.     

Polymerase chain reaction coupling with magnetic nanoparticles-based biotin-avidin system for amplification of chemiluminescent detection signals of nucleic acid

A novel method was established through the detection of chemiluminescent signals of nucleic acid hybridization based on magnetic nanoparticles (MNPs) and PCR. 5' amino- modified specific probes were immobilized on the surface of silanized MNPs by Schiff reaction between amino and aldehyde group. The probes were used to capture the synthetic biotin-dUTP-labeled DNA fragments which were obtained by polymerase chain reaction (PCR). Then these complexes were bonded with streptavidin-modified alkaline phosphatase (SA-AP). Finally the chemiluminescent signals were detected by adding 3-(2'-spiroadamantane)- 4-methoxy -4-(3"-phosphoryloxy) phenyl-1, 2-dioxetane (AMPPD) which was the substrate reagent of AP. The concentration of probes which were immobilized on the surface of MNPs was studied, how to reduce the adsorption of SA-AP on the surface of MNPs was also researched. It was shown that 12.5 pmol of probes were immobilized on 1 mg of MNPs. Aldehyde-MNPs modified with probes could adsorb SA-AP, affecting the sensitivity of chemiluminescene consequently. Reduction of aldehyde group by sodium borohydride and blocking the bare position of MNPs with bovine serum albumin (BSA) could decrease the background of chemiluminescence, and this method has good specificity in detection of chloramphenicol acetyltransferase (CAT) gene.     

Functionalization of electrospun TiO2 nanofibers with Pt nanoparticles and nanowires for catalytic applications

This paper reports a simple procedure for derivatizing the surface of anatase TiO2 nanofibers with Pt nanoparticles and then Pt nanowires. The nanofibers were prepared in the form of a nonwoven mat by electrospinning with a solution containing both poly(vinyl pyrrolidone) and titanium tetraisopropoxide, followed by calcination in air at 510 degrees C. The fiber mat was then immersed in a polyol reduction bath to coat the surface of anatase fibers with Pt nanoparticles of 2-5 nm in size with controllable density of coverage. Furthermore, the coated fibers could serve as a three-dimensional scaffold upon which Pt nanowires of roughly 7 nm in diameter could be grown at a high density and with a length up to 125 nm. The fiber membranes functionalized with Pt nanoparticles and nanowires are interesting for a number of catalytic applications. It was found to show excellent catalytic activity for the hydrogenation of azo bonds in methyl red, which could be operated in a continuous mode by passing the dye solution through the membrane at a flow rate of 0.5 mL/s.     

Exploration of functionalized CdTe nanoparticles for latent fingerprint detection

The potential of using water-soluble photoluminescent nanoparticles of different sizes for latent fingerprint detection has been explored. In this pilot study, green (582 nm) and red (755 nm) CdTe nanocrystals coated with thioglycolic acid were used. Latent fingerprints on aluminum and glass surfaces were successfully labeled with these nanoparticles for time periods ranging from 30 min to 24 h. The labeling is probably due to the amidation reaction between the surface carboxylic groups of the nanoparticles with the amine groups of the biomaterials present in the fingerprint residues. The 582 nm emitting nanoparticles appeared to better label the fingerprint ridges than did the 755 nm emitting ones for both surfaces. However, the 755 nm emitting nanoparticles were able to target the sweat pores within the ridges of the fingerprints. Other than high quantum yield and photostability, the tunable emission wavelength, the narrow bandwidth, the customizable surface characteristics, and the relatively long fluorescence decay lifetime of these nanoparticles are useful and necessary features for the future development of ultra-sensitive, target-specific, background suppressed latent fingerprint detection for forensic applications.     

Functionalization of silica nanoparticles for nucleic acid delivery

Silica nanoparticles (SiNPs) have been widely engineered for biomedical applications, such as bioimaging and drug delivery, because of their high tunability, which allows them to perform specific functions. In this review, we discuss the functionalization and performance of SiNPs for nucleic acid delivery. Nucleic acids, including plasmid DNA (pDNA) and small interfering RNA (siRNA), constitute the next generation molecular drugs for the treatment of intractable diseases. However, their low bioavailability requires delivery systems that can circumvent nuclease attack and kidney filtration to ensure efficient access to the target cell cytoplasm or nucleus. First, we discussed the biological significance of nucleic acids and the parameters required for their successful delivery. Next, we reviewed SiNP designing for nucleic acid delivery with respect to nucleic acid loading and release, cellular uptake, endosomal escape, and biocompatibility. In addition, we discussed the co-delivery potential of SiNPs. Finally, we analyzed the current challenges and future directions of SiNPs for advanced nucleic acid delivery.     

Integration of on-chip isotachophoresis and functionalized hydrogels for enhanced-sensitivity nucleic Acid detection

We introduce an on-chip electrokinetic assay to perform high-sensitivity nucleic acid (NA) detection. This assay integrates electrokinetic sample focusing using isotachophoresis (ITP) with a background signal-removal strategy that employs photopatterened, DNA-functionalized hydrogels. In this multistage assay, ITP first enhances hybridization kinetics between target NAs and end-labeled complementary reporters. After enhanced hybridization, migration through a DNA-functionalized hydrogel region removes excess reporters through affinity interactions. We demonstrate our assay on microRNAs, an important class of low-abundance biomarkers. The assay exhibits 4 orders of magnitude dynamic range, near 1 pM detection limits starting from less than 100 fg of microRNA, and high selectivity for mature microRNA sequences, all within a 10 min run time. This new microfluidic framework provides a unique quantitative assay for NA detection.     

MoS2 nanosheet functionalized with Cu nanoparticles and its application for glucose detection

For the first time, Cu nanoparticles were evenly decorated on MoS₂ nanosheet by chemical reduction. The as-prepared Cu-MoS₂ hybrid was characterized by atomic force microscope (AFM), Raman spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD) and then used to fabricate a non-enzymatic glucose sensor. The performance of our sensor was investigated by cyclic voltammetry and amperometric measurement in alkaline media. Electrochemical tests showed that Cu-MoS₂ hybrid exhibited synergistic electrocatalytic activity on the oxidation of glucose with a high sensitivity of 1055 μA mM⁻¹ cm⁻² and a linear range up to 4 mM.     

Immobilization-free sequence-specific electrochemical detection of DNA using ferrocene-labeled peptide nucleic acid

An electrochemical method for sequence-specific detection of DNA without solid-phase probe immobilization is reported. This detection scheme starts with a solution-phase hybridization of ferrocene-labeled peptide nucleic acid (Fc-PNA) and its complementary DNA (cDNA) sequence, followed by the electrochemical transduction of Fc-PNA-DNA hybrid on indium tin oxide (ITO)-based substrates. On the bare ITO electrode, the negatively charged Fc-PNA-DNA hybrid exhibits a much reduced electrochemical signal than that of the neutral-charge Fc-PNA. This is attributed to the electrostatic repulsion between the negatively charged ITO surface and the negatively charged DNA, hindering the access of Fc-PNA-DNA to the electrode. On the contrary, when the transduction measurement is done on the ITO electrode coated with a positively charged poly(allylamine hydrochloride) (PAH) layer, the electrostatic attraction between the (+) PAH surface and the (-) Fc-PNA-DNA hybrid leads to a much higher electrochemical signal than that of the Fc-PNA. The measured electrochemical signal is proportional to the amount of cDNA present. In terms of detection sensitivity, the PAH-modified ITO platform was found to be more sensitive (with a detection limit of 40 fmol) than the bare ITO counterpart (with a detection limit of 500 fmol). At elevated temperatures, this method was able to distinguish fully matched target DNA from DNA with partial mismatches. Unpurified PCR amplicons were detected using a similar format with a detection limit down to 4.17 amol. This detection method holds great promise for single-base mismatch detection as well as electrochemistry-based detection of post-PCR products.     

Carbon nanocapsules as an effective sensing platform for fluorescence-enhanced nucleic acid detection

In this communication, we demonstrate the proof of concept that carbon nanocapsules (CNCs) can be used as an effective fluorescent sensing platform for nucleic acid detection with selectivity down to single-base mismatch. The detection is accomplished by two steps: (1) CNC adsorbs and quenches the fluorescence of the dye-labeled single-stranded DNA (ssDNA) probe; (2) in the presence of the target, a hybridization event occurs, which produces a double-stranded DNA (dsDNA) that detaches from the CNC surface, leading to recovery of the dye fluorescence.     

Carbon fluoroxide nanoparticles as fluorescent labels and sonosensitizers for theranostic applications

Abstract

Carbon fluoroxide (CFO) nanoparticles (NPs) produced from silicon carbide wafers are used as both fluorescent probes and sonosensitizers for theranostic application. In vitro cell tests were carried out to investigate the feasibility of ultrasound-based therapy with the use of the CFO NPs. The NPs that penetrated inside the cells were shown to provoke cell destruction after application of an ultrasound treatment. No significant toxic effect was observed when the cells were treated with NP concentrations up to 0.5 mg ml^?1 without applying ultrasound treatment. The obtained results open a new way toward cancer therapy strategies.     

Carbon fluoroxide nanoparticles as fluorescent labels and sonosensitizers for theranostic applications

Carbon fluoroxide (CFO) nanoparticles (NPs) produced from silicon carbide wafers are used as both fluorescent probes and sonosensitizers for theranostic application. In vitro cell tests were carried out to investigate the feasibility of ultrasound-based therapy with the use of the CFO NPs. The NPs that penetrated inside the cells were shown to provoke cell destruction after application of an ultrasound treatment. No significant toxic effect was observed when the cells were treated with NP concentrations up to 0.5 mg ml⁻¹ without applying ultrasound treatment. The obtained results open a new way toward cancer therapy strategies.     

Luminescent lanthanide nanoparticles as labels in DNA microarrays for quantification of methyl tertiary butyl ether degrading bacteria

We report application of lanthanide nanoparticles for DNA quantification in a microarray platform as a substitute for conventional organic fluorophores. A non-PCR based DNA microarray assay for quantifying bacteria capable of biodegrading methyl tertiary-butyl ether (MTBE) was demonstrated. Probe DNA was immobilized on a glass surface, hybridized with biotinylated target DNA and subsequently incubated with Neutravidin-biofunctionalized nanoparticles. The fluorescence spot intensities, measured by a commercial laser scanner, show a linear relationship (R2 = 0.98) with bacterial 16S rDNA over a range of target DNA concentrations, while the background fluorescence remained low. In addition, nanoparticles fluorescence shows a stronger intensity than Quasar570 (Cy3). Present sensitivity of the assay is 10 pM of target DNA. The selectivity of the DNA-nanoparticle-probes to discriminate a non-target DNA with two base pairs mismatch in the 16S rDNA gene sequence was shown. The use of Eu:Gd2O3 nanoparticles as biolabels provides a relatively non-toxic, inexpensive, rapid and sensitive alternative to the materials currently used in DNA microarrays.     

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.     

Pd nanowires as new biosensing materials for magnified fluorescent detection of nucleic acid

The designed synthesis of new nanomaterials with controlled shape, composition, and structure is critical for tuning their physical and chemical properties, and further developing interesting analytical sensing devices. Herein, we presented that Pd nanowires (NWs) can be used as a new biosensing platform for high-sensitivity nucleic acid detection. The general sensing concept is based on the fact that Pd NWs can adsorb the fluorescently labeled single-stranded DNA probe and lead to substantial fluorescence quenching of dye, followed by specific hybridization with the complementary region of the target DNA sequence. This results in desorption of double-stranded DNA from Pd NWs surface and subsequent recovery of fluorescence. Furthermore, an amplification strategy based on Pd NWs for nucleic acid detection by using exonuclease III (Exo III) was demonstrated. The present dual-magnification sensing system combined Pd NWs with Exo III has a detection range of 1.0 nM to 2.0 μM with the detection limit of 0.3 nM (S/N = 3), which is about 20-fold higher than that of traditional unamplified homogeneous assays.