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.

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.

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.

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.

Ultrasensitive electrochemical supersandwich DNA biosensor using a glassy carbon electrode modified with gold particle-decorated sheets of graphene oxide

We describe a supersandwich type of electrochemical DNA biosensor based on the use of a glassy carbon electrode (GCE) modified with reduced graphene oxide (rGO) sheets that are decorated with gold nanoparticles (Au NPs). Thiolated capture DNA (probe DNA) was covalently linked to the Au NPs on the surface of the modified GCE via formation of Au-S bonds. In presence of target DNA, its 3′ terminus hybridizes with capture probe and the 5′ terminus hybridizes with signal probe labeled with Methylene Blue (MB). On increasing the concentration of target DNA, hybridization between signal probe and target DNA results in the formation of three different DNA sequences that form a supersandwich structure. The signal intensity of MB improves distinctly with increasing concentrations of target DNA in the sample solution. The assembling process on the surface of the electrode was studied by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). Differential pulse voltammetry (DPV) was used to monitor the hybridization event by measuring the changes in the peak current for MB. Under optimal conditions, the peak currents in DPV for MB linearly increase with the logarithm of target DNA concentration in the range from 0.1 μM to1.0 fM, with a detection limit of 0.35 fM (at an signal/noise ratio of 3). This biosensor exhibits good selectivity, even over single-base mismatched target DNA.

Glucose biosensor based on a platinum electrode modified with rhodium nanoparticles and with glucose oxidase immobilized on gold nanoparticles

We have developed an enzymatic glucose biosensor that is based on a flat platinum electrode which was covered with electrophoretically deposited rhodium (Rh) nanoparticles and then sintered to form a large surface area. The biosensor was obtained by depositing glucose oxidase (GOx), Nafion, and gold nanoparticles (AuNPs) on the Rh electrode. The electrical potential and the fractions of Nafion and GOx were optimized. The resulting biosensor has a very high sensitivity (68.1 μA mM^?1 cm^?2) and good linearity in the range from 0.05 to 15 mM (r?=?0.989). The limit of detection is as low as 0.03 mM (at an SNR of 3). The glucose biosensor also is quite selective and is not interfered by electroactive substances including ascorbic acid, uric acid and acetaminophen. The lifespan is up to 90 days. It was applied to the determination of glucose in blood serum, and the results compare very well with those obtained with a clinical analyzer.

Novel glucose biosensor based on a glassy carbon electrode modified with hollow gold nanoparticles and glucose oxidase

A novel glucose biosensor is presented as that based on a glassy carbon electrode modified with hollow gold nanoparticles (HGNs) and glucose oxidase. The sensor exhibits a better differential pulse voltammetric response towards glucose than the one based on conventional gold nanoparticles of the same size. This is attributed to the good biological conductivity and biocompatibility of HGNs. Under the optimal conditions, the sensor displays a linear range from 2.0?×?10^?6 to 4.6?×?10^?5?M of glucose, with a detection limit of 1.6?×?10^?6?M (S/N?=?3). Good reproducibility, stability and no interference make this biosensor applicable to the determination of glucose in samples such as sports drinks.

Amperometric hydrogen peroxide biosensor based on a glassy carbon electrode modified with polythionine and gold nanoparticles

We report on a novel hydrogen peroxide biosensor that was fabricated by the layer-by-layer deposition method. Thionine was first deposited on a glassy carbon electrode by two-step electropolymerization to form a positively charged surface. The negatively charged gold nanoparticles and positively charged horseradish peroxidase were then immobilized onto the electrode via electrostatic adsorption. The sequential deposition process was characterized using electrochemical impedance spectroscopy by monitoring the impedance change of the electrode surface during the construction process. The electrochemical behaviour of the modified electrode and its response to hydrogen peroxide were studied by cyclic voltammetry. The effects of the experimental variables on the amperometric determination of H₂O₂ such as solution pH and applied potential were investigated for optimum analytical performance. Under the optimized conditions, the biosensor exhibited linear response to H₂O₂ in the concentration ranges from 0.20 to 1.6?mM and 1.6 to 4.0?mM, with a detection limit of 0.067?mM (at an S/N of 3). In addition, the stability and reproducibility of this biosensor was also evaluated and gave satisfactory results.

Label-free and ultrasensitive electrochemiluminescence detection of microRNA based on long-range self-assembled DNA nanostructures

Electrochemiluminescence (ECL) integrates the advantages of electrochemical detection and chemiluminescent techniques. The method has received particular attention because it is highly sensitive and selective, has a wide linear range but low reagent costs. The use of nanomaterials with their unique physical and chemical properties has led to new kinds of biosensors that exhibit high sensitivity and stability. Compared to other nanomaterials, DNA nanostructures are more biocompatible, more hydrophilic, and thus less prone to nonspecific adsorption onto the electrode surface. We describe here a label-free and ultrasensitive ECL biosensor for detecting a cancer-associated microRNA at a femtomolar level. We have designed two auxiliary probes that cause the formation of a long-range self-assembly in the form of a μm-long 1-dimensional DNA concatamer. These can be used as carriers for signal amplification. The intercalation of the ECL probe Ru(phen)₃ ²⁺ into the grooves of the concatamers leads to a substantial increase in ECL intensity. This amplified sensor shows high selectivity for discriminating complementary target and other mismatched RNAs. The biosensor enables the quantification of the expression of microRNA-21 in MCF-7 cells. It also displays very low limits of detection and provides an alternative approach for the detection of RNA or DNA detection in diagnostics and gene analysis.

Label-free electrochemical DNA biosensor based on a glassy carbon electrode modified with gold nanoparticles, polythionine, and graphene

We describe the fabrication of a sensitive label-free electrochemical biosensor for the determination of sequence-specific target DNA. It is based on a glassy carbon electrode (GCE) modified with graphene, gold nanoparticles (Au-NPs), and polythionine (pThion). Thionine was firstly electropolymerized on the surface of the GCE that was modified with graphene by cyclic voltammetry. The Au-NPs were subsequently deposited on the surface of the pThion/graphene composite film by adsorption. Scanning electron microscopy and electrochemical methods were used to investigate the assembly process. Differential pulse voltammetry was employed to monitor the hybridization of DNA by measuring the changes in the peak current of pThion. Under optimal conditions, the decline of the peak current is linearly related to the logarithm of the concentration of the target DNA in the range from 0.1 pM to 10 nM, with a detection limit of 35 fM (at an S/N of 3). The biosensor exhibits good selectivity, acceptable stability and reproducibility.

Sensitive detection of hydrogen peroxide in foodstuff using an organic�Cinorganic hybrid multilayer-functionalized graphene biosensing platform

We report on a new electrochemical biosensing strategy for the sensitive detection of hydrogen peroxide (H₂O₂) in foodstuff samples. It is based on a gold electrode modified with layer of graphene patterned with a multilayer made from an organic?Cinorganic hybrid nanomaterial. Initially, a layer of thionine (Th) was assembled on the surface of the graphene nanosheets, and these were then cast on the surface of the electrode for the alternate assembly of gold nanoparticles and horseradish peroxidase. The large surface-to-volume ratio and high conductivity of the nanosheets provides a benign microenvironment for the construction of the biosensor. The use of such a multilayer not only shortens the electron transfer pathway of the active center of the enzyme due to the presence of gold nanoparticles, but also enhances the electrocatalytic efficiency of the biosensor toward the reduction of H₂O₂. The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. The number of layers, the operating potential, and the pH of the supporting electrolyte were optimized. Linear response is obtained for the range from 0.5???M to 1.8?mM of H₂O₂, the detection limit is 10 nM (at S/N?=?3), and 95% of the steady-state current is reached within 2?s. The method was applied to sense H₂O₂ in spiked sterilized milk and correlated excellently with the permanganate titration method.

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.

Sensitive fluorescent detection of Staphylococcus aureus using nanogold linked CdTe nanocrystals as signal amplification labels

A sensitive fluorescent assay was developed for the detection of DNA specifically for Staphylococcus aureus. A sandwich-type detection system was fabricated by first immobilizing biotinylated capture DNA on avidin-modified wells of microplates, then hybridizing the capture DNA with one end of the target DNA, and then recognizing the other end of the target DNA with a signal probe labeled with CdTe nanocrystals and gold nanoparticles (Au-NPs) at the 3′- and 5′-terminus, respectively. Hybridization was monitored by measuring the fluorescent intensity of the assembly. The experimental results demonstrated that the incorporation of Au-NPs in this detection system can significantly enhance the sensitivity and the selectivity because a single Au-NP can be loaded with hundreds of signal DNA probe strands modified with CdTe nanocrystals. Under the optimized conditions, a detection limit of 10 fmol of DNA per L can be achieved and at least 50 colony forming units of Staph. aureus per mL of sample can be detected. The method was assessed by analyzing real samples, and it was validated by comparing it to an official standard method.

A DNA biosensor based on resonance light scattering using unmodified gold bipyramids

We report on a novel biosensor for determining sequence-specific DNA. It is based on resonance light scattering (RLS) caused by the aggregation of gold bipyramids. These display localized surface plasmon resonance and can be used as a bioprobe. The absorption spectra and the transmission electron micrographs provide visual evidence of the aggregation of the gold bipyramids in the presence of DNA. The RLS intensity of the gold bipyramids increases with the concentration of the target DNA. The method was successfully applied to the determination of a 30-mer single-stranded oligonucleotide and works over the 0.1–10?nM concentration range.

Determination of organophosphate pesticides using an acetylcholinesterase-based biosensor based on a boron-doped diamond electrode modified with gold nanoparticles and carbon spheres

We report on a biosensor for organophosphate pesticides (OPs) by exploiting their inhibitory effect on the activity of acetylcholinesterase (AChE). A boron-doped diamond (BDD) electrode was modified with a nanocomposite prepared from carbon spheres (CSs; with an average diameter of 500 nm) that were synthesized from resorcinol and formaldehyde, and then were coated with gold nanoparticles (AuNPs) by chemically growing them of the CSs. Compared to a bare BDD electrode, the electron transfer resistance is lower on this new electrode. Compared to an electrode without Au-NPs, the peak potential is negatively shifted by 42 mV, and the peak current is increased by 55 %. This is ascribed to the larger surface in the AuNP-CS nanocomposite which improves the adsorption of AChE, enhances its activity, and facilitates electrocatalysis. Under optimum conditions, the inhibitory effect of chlorpyrifos is linearly related to the negative log of its concentration in the 10^?11 to 10^?7 M range, with a detection limit of 1.3?×?10^?13 M. For methyl parathion, the inhibition effect is linear in the 10^?12 to 10^?6 M range, and the detection limit is 4.9?×?10^?13 M. The biosensor exhibits good precision and acceptable operational and temporal stability.

A glassy carbon electrode modified with graphene and tyrosinase immobilized on platinum nanoparticles for sensing organophosphorus pesticides

An amperometric biosensor is described for the detection of organophosphorus pesticides. It is based on the enzyme tyrosinase immobilized on platinum nanoparticles and the use of a glassy carbon electrode modified with graphene. Tyrosinase was immobilized on the electrode surface via electrostatic interaction between a monolayer of cysteamine and the enzyme. In the presence of catechol as a substrate, the pesticides chlorpyrifos, profenofos and malathion can be determined as a result of their inhibition of the enzyme which catalyzes the oxidation of catechol to o-quinone. Platinum nanoparticles and graphene effectively enhance the efficiency of the electrochemical reduction of o-quinone, thus improving sensitivity. Under optimum experimental conditions, the inhibition effect of the pesticides investigated is proportional to their concentrations in the lower ppb-range. The detection limits are 0.2, 0.8 and 3?ppb for chlorpyrifos, profenofos and malathion, respectively. The biosensor displays good repeatability and acceptable stability.

Simple construction of an enzymatic glucose biosensor based on a nanocomposite film prepared in one step from iron oxide, gold nanoparticles, and chitosan

The one-step synthesis is reported of a nanofilm composed of iron oxide and gold nanoparticles in a chitosan matrix that can act as a novel matrix for the immobilization of glucose oxidase (GOx) to fabricate a glucose biosensor. The use for the composite film strongly increased the effective electrode surface for loading of GOx. The size and shape of the iron oxide nanoparticles were examined by transmission electron micrograph. Direct electron transfer and electrocatalysis by GOx was investigated via cyclic voltammetry and chronoamperometry. Under optimized conditions, the biosensor has a response time of 6?s and a linear response in the range between 3???M and 0.57?mM of glucose, with a detection limit of 1.2???M at a signal-to-noise ratio of 3. This novel and disposable mediatorless glucose biosensor may form the basis for a future mass-produced glucose biosensor.

Gold electrodes modified with gold nanoparticles and thio compounds for electrochemical sensing of dopamine alone and in presence of potential interferents. A comparative study

Gold electrodes were modified with self assembled layers (SAMs) composed of mercaptopropionic acid, thiodipropionic acid, dithiodipropionic acid, cysteamine and gold nanoparticles and used to study the electrooxidation of dopamine (DA) in solution at pH 7. SAMs endowed with gold nanoparticles gave the highest catalytic effect. The results showed that such electrodes are capable of resolving the oxidation peaks of DA, ascorbic acid, and uric acid which is most favourable with respect to the detection of DA in physiological matrices.

Luminescent Ru(bpy)3 2+-doped silica nanoparticles for imaging of intracellular temperature

Silica nanoparticles doped with the luminescent temperature probe Ru(bpy)₃ ²⁺ were prepared by a modified Stöber method and are shown to enable optical sensing of intracellular temperatures. Based on the regrowth of silica nanoseeds, the ruthenium probe was easily incorporated and then covered with a shell of pure silica. The resulting nanothermometers were immune to the quenching by oxygen owing to the outer silica layer. The nanoparticles were further coated with poly-L-lysine in order to reduce cytotoxicity and to warrant cellular uptake. The luminescence of these nanosensors is rather sensitive to temperature in the physiological range (25–45 °C), with a decrease of ?1.26 % in intensity per °C increase in temperature. The nanosensors were internalized into living cells of a hepatocellular carcinoma cell line along with gold nanorods. These display longitudinal surface plasmon resonance absorption at ~808 nm that causes a local rise in temperature. The microscopically captured luminescence intensity of the nanosensors after 808 nm irradiation of the gold nanorods decayed with increasing temperature, thereby indicating successful imaging of temperature.

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.