Detection of DNA hybridization by various spectroscopic methods using the copper tetraphenylporphyrin complex as a probe

We are presenting new and highly sensitive hybridization assays. They are based on various spectroscopic methods including resonance light scattering, circular dichroism, ultraviolet spectra and fluorescence spectra, as well as atomic force microscopy, and relies on the interaction of the Cu(II), Ni(II), Mg(II), Co(II), Cd(II), and Zn(II) complexes, respectively, of tetraphenylporphyrin (TPP) with double-strand DNA (dsDNA) and single strand DNA (ssDNA). The interaction results in amplified resonance light scattering (RLS) signals and enables the detection of hybridization without the need for labeling DNA. The RLS signals are strongest in case of the Cu (II)-TPP complex which therefore was selected as the probe. The technique is simple, robust, accurate, and can be completed in less than one hour.

Colorimetric detection of peroxynitrite-induced DNA damage using gold nanoparticles, and on the scavenging effects of antioxidants

We describe a rapid and convenient colorimetric method for the detection of oxidative DNA damage caused by peroxynitrite (ONOO^?) using unmodified gold nanoparticles (AuNPs). AuNPs are stable in the presence of single-stranded DNA (ssDNA) against the aggregation induced by a high ionic strength. If adsorbed ssDNA are cleaved by ONOO^? to form smaller fragments, the AuNPs rapidly aggregate due to electrostatic attraction. As a result, the color of the solution changes from red to blue, and this can be seen with bare eyes. We also have evaluated the activity of the antioxidants gallic acid, ascorbic acid and caffeic acid to scavenge ONOO^?. This method therefore also can be applied to screen for anti-oxidation drugs and agents.

Ultra-sensitive electrochemical DNA biosensor based on signal amplification using gold nanoparticles modified with molybdenum disulfide,graphene and horseradish peroxidase

We have developed an ultra-sensitive electrochemical DNA biosensor by assembling probe ssDNA on a glassy carbon electrode modified with a composite made from molybdenum disulfide, graphene, chitosan and gold nanoparticles. A thiol-tagged DNA strand coupled to horseradish peroxidase conjugated to AuNP served as a tracer. The nanocomposite on the surface acts as relatively good electrical conductor for accelerating the electron transfer, while the enzyme tagged gold nanoparticles provide signal amplification. Hybridization with the target DNA was studied by measuring the electrochemical signal response of horseradish peroxidase using differential pulse voltammetry. The calibration plot is linear in the 5.0?×?10^?14 and 5.0?×?10^?9 M concentration range, and the limit of detection is 2.2?×?10^?15 M. The biosensor displays high selectivity and can differentiate between single-base mismatched and three-base mismatched sequences of DNA. The approach is deemed to provide a sensitive and reliable tool for highly specific detection of DNA.

Improving surface plasmon resonance imaging of DNA by creating new gold and silver based surface nanostructures

The use of nanoparticles (NPs) can substantially improve the analytical performance of surface plasmon resonance imaging (SPRi) in general, and in DNA sensing in particular. In this work, we report on the modification of the gold surface of commercial biochips with gold nanospheres, silica-coated gold nanoshells, and silver nanoprisms, respectively. The NPs were tethered onto the surface of the chip and functionalized with a DNA probe. The effects of tethering conditions and varying nanostructures on the SPRi signals were evaluated via hybridization assays. The results showed that coupling between planar surface plasmons and electric fields, generated by localized surface plasmons of the NPs, is mandatory for signal enhancement. Silver nanoprisms gave the best results in improving the signal change at a target DNA concentration of <50 nM by +50 % (compared to a conventional SPRi chip). The limit of detection for the target DNA was 0.5 nM which is 5 times less than in conventional SPRi.

Immunoassay for serum alpha-fetoprotein using silver nanoparticles and detection via resonance light scattering

The assay for alpha-fetoprotein (AFP) is based on the use of immobilized anti-AFP labeled with silver nanoparticles (AgNPs). The immunoreaction between the labeled antibody against AFP and free AFP takes place in pH 6.0 solution and leads to the formation of the respective immunocomplex which displays enhanced resonance light scattering (RLS) intensity at 480 nm. Under the optimal conditions, the intensity of the enhanced RLS is proportional to the concentration of AFP in the range from 0.10 to 50 ng mL^?1, with a detection limit of 40 pg mL^?1. The characteristics of RLS, the immunocomplex, the immuno response, and the optimum conditions of the immunoreaction have been investigated. The concentration of AFP in 20 serum specimens was determined by the new assay, and results are consistent with those obtained with a commercially available ELISA kit.

Streptavidin-enhanced surface plasmon resonance biosensor for highly sensitive and specific detection of microRNA

We are presenting a method for sensitive and specific detection of microRNA (miRNA) using surface plasmon resonance. A thiolated capture DNA probe with a short complete complementary sequence was immobilized on the gold surface of the sensor to recognize the part sequence of target miRNA, and then an oligonucleotide probe linked to streptavidin was employed to bind the another section of the target. The use of the streptavidin-oligonucleotide complex caused a ~5-fold increase in signal, improved the detection sensitivity by a factor of ~24, and lowered the detection limit to 1.7 fmol of miR-122. This specificity allowed a single mismatch in the target miRNA to be discriminated. The whole assay takes 30 min, and the surface of the sensor can be regenerated at least 30 times without loss in performance. The method was successfully applied to the determination of miRNA spiked into human total RNA samples.

Sensitive DNA-hybridization biosensors based on gold nanoparticles for testing DNA damage by Cd(II) ions

A DNA biosensor was constructed by immobilizing a 20-mer oligonucleotide probe and hybridizing it with its complementary oligomer on the surface of a glassy carbon electrode modified with gold nanoparticles. The properties of the biosensor and its capability of recognizing its complementary sequence were studied by electrochemical impedance spectroscopy. The oxidative stress caused by cadmium ions can be monitored by differential pulse voltammetry using the cobalt(III)tris(1,10-phenanthroline) complex and methylene blue as electrochemical indicators. The biosensor is capable of indicating damage caused by Cd(II) ions in pH 6.0 solution. The results showed that the biosensor can be used for rapid screening for DNA damage.

Chronocoulometric DNA biosensor based on a glassy carbon electrode modified with gold nanoparticles, poly(dopamine) and carbon nanotubes

We describe a sensitive chronocoulometric biosensor for the sequence-specific detection of DNA. It is based on a glassy carbon electrode modified with multi-walled carbon nanotubes, polydopamine, and gold nanoparticles. The ruthenium(III)hexammine complex acts as the electrochemical indicator. Electrochemical impedance spectra and scanning electron microscopy are employed to investigate the assembly of the electrode surface. The signals of the ruthenium complex electrostatically bound to the anionic phospho groups of the DNA strands are measured by chronocoulometry before and after hybridization. The difference in signal intensity is linearly related to the logarithm of the concentration of the target DNA in the range of 1.0 nM to 10 fM with a detection limit of 3.5fM (S/N?=?3) under optimal conditions. This biosensor exhibits excellent sensitivity and selectivity and has been used for an assay of complementary target DNA in human serum sample with satisfactory results.

Investigation on the interaction between anthracyclines and DNA in the presence of paclitaxel by resonance light scattering technique

We have developed a method to investigate the interaction between DNA-targeted anthracyclines and DNA in the presence of the drug paclitaxel. It is based on resonance light scattering (RLS) and on the finding that anthracyclines when bound to DNA undergo a dramatic enhancement in their RLS intensities, while paclitaxel does not display such an effect. However, the RLS intensities of the anthracyclines-DNA associates are remarkably enhanced again on addition of paclitaxel. UV-visible spectra reveal interactions between paclitaxel and anthracyclines, but no reaction between paclitaxel and DNA. Consequently, paclitaxel, though not DNA-targeted, can improve the DNA-binding capabilities of anthracyclines. Binding constants between anthracyclines and DNA, and improved efficiency of paclitaxel on the DNA-binding capabilities of anthracyclines were calculated. The DNA binding constants of doxorubicin, epirubicin, and mitoxantrone, respectively, are 4.53?×?10⁵?L?mol^?1, 6.05?×?10⁵?L?mol^?1, and 9.47?×?10⁵?L?mol^?1. The improved values in presence of paclitaxel are 78%, 47% and 19%. We also have investigated the effects of drug concentrations and the order of adding the drugs. Displacement studies (using methylene blue as a competitive agent) provided additional information on the mechanisms of the interaction between paclitaxel and anthracyclines.

CdS quantum dots as a fluorescent sensing platform for nucleic acid detection

We demonstrate that CdS quantum dots (QDs) can be applied to fluorescence-enhanced detection of nucleic acids in a two-step protocol. In step one, a fluorescently labeled single-stranded DNA probe is adsorbed on the QDs to quench its luminescence. In step two, the hybridization of the probe with its target ssDNA produces a double-stranded DNA which detaches from the QD. This, in turn, leads to the recovery of the fluorescence of the label. The lower detection limit of the assay is as low as 1?nM. The scheme (that was applied to detect a target DNA related to the HIV) is simple and can differentiate between perfectly complementary targets and mismatches.

Dendrimer-based biosensor for chemiluminescent detection of DNA hybridization

We report on a highly sensitive chemiluminescent (CL) biosensor for the sequenc-specific detection of DNA using a novel bio barcode DNA probe modified with gold nanoparticles that were covered with a dendrimer. The modified probe is composed of gold nanoparticles, a dendrimer, the CL reagent, and the DNA. The capture probe DNA was immobilized on magnetic beads covered with gold. It first hybridizes with the target DNA and then with one terminal end of the signal DNA on the barcoded DNA probe. CL was generated by adding H₂O₂ and Co(II) ions as the catalyst. The immobilization of dendrimer onto the gold nanoparticles can significantly enhance sensitivity and gives a detection limit of 6 fmol L^-1 of target DNA.

Highly sensitive determination of doxorubicin and daunorubicin based on their effect on the resonance light scattering signals of the ethidium-DNA complex

We have developed a resonance light scattering (RLS) quenching assay for the highly sensitive determination of doxorubicin (DOX) and daunorubicin (DAU). It is based on the reduction of the intensity of the shoulder of the RLS spectra at 443?nm. The intensity of the RLS of the ethidium-DNA system decrease linearly on addition of trace quantities of DOX or DAU within the concentration range of 0.008 to 12.0???g?mL^?1 for DOX, and of 0.010 to 21.0???g?mL^?1 for DAU. The detection limits are 3.0 and 5.0?ng?mL^?1, respectively. The assay was successfully applied to the determination of DAU in synthetic and serum samples. Compared to the reported methods for anthracyclines, this assay displays higher sensitivity, lower detection limits, and a wider linear range.

Molecular beacon based biosensor for the sequence-specific detection of DNA using DNA-capped gold nanoparticles-streptavidin conjugates for signal amplification

We describe a highly sensitive and selective molecular beacon-based electrochemical impedance biosensor for the sequence-specific detection of DNA. DNA-capped conjugates between gold nanoparticles (Au-NPs) and streptavidin are used for signal amplification. The molecular beacon was labeled with a thiol at its 5′ end and with biotin at its 3′ end, and then immobilized on the surface of a bare gold electrode through the formation of Au-S bonds. Initially, the molecular beacon is present in the “closed” state, and this shields the biotin from being approached by streptavidin due to steric hindrance. In the presence of the target DNA, the target DNA molecules hybridize with the loop and cause a conformational change that moves the biotin away from the surface of the electrode. The biotin thereby becomes accessible for the reporter (the DNA-streptavidin capped Au-NPs), and this results in a distinct increase in electron transfer resistance. Under optimal conditions, the increase in resistance is linearly related to the logarithm of the concentration of complementary target DNA in the range from 1.0 fM to 0.1 μM, with a detection limit of 0.35 fM (at an S/N of 3). This biosensor exhibits good selectivity, and acceptable stability and reproducibility.

Fluorescence energy transfer-based multiplexed hybridization assay using gold nanoparticles and quantum dot conjugates on photonic crystal beads

A multiplexed assay strategy was developed for the detection of nucleic acid hybridization. It is based on fluorescence resonance energy transfer (FRET) between gold nanoparticles (AuNPs) and multi-sized quantum dots (QDs) deposited on the surface of silica photonic crystal beads (SPCBs). The SPCBs were first coated with a three-layer primer film formed by the alternating adsorption of poly(allylamine hydrochloride) and poly(sodium 4-styrensulfonate). Probe DNA sequences were then covalently attached to the carboxy groups at the surface of the QD-coated SPCBs. On addition of DNA-AuNPs and hybridization, the fluorescence of the donor QDs is quenched because of the close proximity of the AuNPs. However, the addition of target DNA causes a recovery of the fluorescence of the QD-coated SPCBs, thus enabling the quantitative assay of hybridized DNA. Compared to fluorescent dyes acting as acceptors, the use of AuNPs results in much higher quenching efficiency. The multiplexed assay displays a wide linear range, high sensitivity, and very little cross-reactivity. This work, where such SPCBs are used for the first time in a FRET assay, is deemed to present a new and viable approach towards high-throughput multiplexed gene assays.

Impedimetric aptasensor for Staphylococcus aureus based on nanocomposite prepared from reduced graphene oxide and gold nanoparticles

We describe here an aptasensor for the ultrasensitive detection of Staphylococcus aureus by electrochemical impedance spectroscopy (EIS). Single-stranded DNA was linked to a nanocomposite prepared from reduced graphene oxide (rGO) and gold nanoparticles (AuNP). Thiolated ssDNA was covalently linked to the AuNPs linked to rGO, and probe DNA was immobilized on the surface of an AuNP-modified glassy carbon electrode to capture and concentrate Staph. aureus. The probe DNA of the aptasensor selectively captures the target bacteria in its three-dimensional space, and these results in a dramatic increase in impedance. Scanning electron microscopy, cyclic voltammetry and EIS were used to monitor the single steps of the electrode assembly process. The effect was utilized to quantify the bacteria in the concentration range from 10 to 10⁶ cfu mL^?1 and with a detection limit of 10 cfu mL^?1 (S/N?=?3). The relative standard deviation of Staphylococcus aureus detection was equal to 4.3 % (10⁵ cfu mL^?1, n?=?7). In addition to its sensitivity, the biosensor exhibits high selectivity over other pathogens.

SERS-based DNA detection in aqueous solutions using oligonucleotide-modified Ag nanoprisms and gold nanoparticles

Oligonucleotide-modified nanoparticle conjugates show highly promising potential for SERS-based DNA detection. However, it remains challenging to carry out the SERS-based DNA detection in aqueous solutions directly using oligonucleotide-modified nanoparticles, because the Raman reporters would exhibit lower signals when they are dispersed in aqueous solutions than laid on “dry” metal nanoparticles. Here, we synthesized stable oligonucleotide-modified Ag nanoprism conjugates, and performed SERS-based DNA detection in aqueous solution directly by using such conjugates in combination with Raman reporter-labeled, oligonucleotide-modified gold nanoparticles. The experimental results indicate that this SERS-based DNA detection approach exhibited a good linear correlation between SERS signal intensity and the logarithm of target DNA concentration ranging from 10^?11～10^?8 M. This sensitivity is comparable to those SERS-based DNA detection approaches with the “dry” process. Additionally, a similar correlation could also be observed in duplex target DNA detection by SERS hybrid probes. Our results suggest that the oligonucleotide-modified Ag nanoprisms may be developed as a powerful SERS-based DNA detection tool.

Electrochemical DNA biosensor for the detection of Trichoderma harzianum based on a gold electrode modified with a composite membrane made from an ionic liquid, ZnO nanoparticles and chitosan, and by using acridine orange as a redox indicator

An electrochemical DNA biosensor was developed that is based on a gold electrode modified with a nanocomposite membrane made from an ionic liquid, ZnO nanoparticles and chitosan. A single-stranded DNA probe was immobilized on this electrode. Acridine orange was used as the hybridization probe for monitoring the hybridization of the target DNA. The biosensor was capable of detecting target DNA in the concentration range from 1.0?×?10?C14 to 1.8?×?10?C4?mol?L-1, with a detection limit of 1.0?×?10?C15?mol?L-1. The approach towards constructing a DNA biosensor allows studies on the hybridization even with crude DNA fragments and also to analyze sample obtained from real samples. The results show that the DNA biosensor has the potential for sensitive detection of a specific sequence of the Trichoderma harzianum gene and provides a quick, sensitive and convenient method for the study of microorganisms.

Sandwich immunoassay for alpha-fetoprotein in human sera using gold nanoparticle and magnetic bead labels along with resonance Rayleigh scattering readout

We describe a sensitive sandwich immunoassay for alpha-fetoprotein (AFP). It is making use of gold nanoparticles (GNPs) and magnetic beads (MBs) as labels, and of resonance Rayleigh scattering for detection. Two antibodies were labeled with GNPs and MBs, respectively, and MB-antigen-GNP complexes were formed in the presence of antigens. The MB labels also serve as solid phase carriers that can be used to magnetically separate the immuno complex. The GNP labels are used as optical probes, and Rayleigh scattering was used to determine the concentration of free GNPs-antibody after separation of the MB-antigen-GNP complexes. The concentration of AFP is related to the intensity of light scattered by free GNPs in the 13.6 pM to 436 pM concentration range, and the limit of detection is 13.6 pM. The method was applied to the determination of AFP in sera of cancer patients, and the results agree well with those obtained by conventional ELISA.

Immunoassay for SKOV-3 human ovarian carcinoma cells using a graphene oxide-modified electrode

We describe a highly sensitive electrochemical immunoassay for the tumor maker HER2 on the surface of SKOV-3 human ovarian cancer cells. Following the binding of the cancer cells, ssDNA-labeled anti-HER2 antibody (ssDNA-Ab; the detection antibody) was added to conjugate unbound antigen on the target cells. Following hybridization of ssDNA with its complementary DNA, daunorubicin was injected in order to intercalate into the duplex. This enables electron transfer between daunorubicin and electrode to take place. The GO film strongly amplifies the redox signal of daunorubicin. This new assay has a detection limit of 5.2 cells per mL and in our opinion holds great promise for clinical screening of cancer biomarkers and point-of-care diagnostics.