Ultrasensitive flow injection chemiluminescence detection of DNA hybridization using signal DNA probe modified with Au and CuS nanoparticles

A novel and sensitive flow injection chemiluminescence assay for sequence-specific DNA detection based on signal amplification with nanoparticles (NPs) is reported in the present work. The "sandwich-type" DNA biosensor was fabricated with the thiol-functionalized capture DNA first immobilized on an Au electrode and hybridized with one end of target DNA, the other end of which was recognized with a signal DNA probe labeled with CuS NPs and Au NPs on the 3'- and 5'-terminus, respectively. The hybridization events were monitored by the CL intensity of luminol-H2O2-Cu(2+) after the cupric ions were dissolved from the hybrids. We demonstrated that the incorporation of Au NPs in this sensor design significantly enhanced the sensitivity and the selectivity because a single Au NP can be loaded with hundreds of signal DNA probe strands, which were modified with CuS NPs. The ratios of Au NPs, signal DNA probes, and CuS NPs modified on the gold electrode were approximately 1/101/103. A preconcentration process of cupric ions performed by anodic stripping voltammetry technology further increased the sensor performance. As a result of these two combined effects, this DNA sensor could detect as low as femtomolar target DNA and exhibited excellent selectivity against two-base mismatched DNA. Under the optimum conditions, the CL intensity was increased with the increase of the concentration of target DNA in the range of 2.0 x 10(-14)-2.0 x 10(-12) M. A detection limit of 4.8 x 10(-15) M target DNA was achieved.     

Electrochemical biosensor based on multi-walled carbon nanotubes and Au nanoparticles synthesized in chitosan

A new biosensor is prepared by cross-linking glucose oxidase (GOD) with glutaradehyde at the electrode combining Au nanoparticles (AuNP) with multi-walled carbon nanotubes (MWCNTs). Au nanoparticles-doped chitosan (CS) solution (AuNP-CS) is prepared by treating the CS solution followed by chemical reduction of Au (III) with NaBH4. MWCNTs are then dispersed in AuNP-CS solution. TEM, FT-IR, and UV-Vis show that the AuNP-CS solution is highly dispersed and stable. The synergistic effect between AuNP and CNTs of the AuNP-CNTs-CS material has been investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and amperometric methods. The modified glassy carbon electrode (GCE) allows low-potential detection of H2O2 with high sensitivity and fast response time. With the immobilization of GOD, a biosensor has been constructed. In phosphate buffer solutions (PBS, pH 7.0), nearly free interference determination of glucose has been realized at 0.4 V(vs. Ag/AgCl/3.0 M KCI) with a wide linear range from 2.0 x 10(-5) to 1.5 x 10(-2) M and a fast response time within 5s. The biosensor has been used to determine glucose in human serum samples and the results are satisfactory.     

Detection of adenosine triphosphate with an aptamer biosensor based on surface-enhanced Raman scattering

A simple, ultrasensitive, highly selective, and reagent-free aptamer-based biosensor has been developed for quantitative detection of adenosine triphosphate (ATP) using surface-enhanced Raman scattering (SERS). The sensor contains a SERS probe made of gold nanostar@Raman label@SiO(2) core-shell nanoparticles in which the Raman label (malachite green isothiocyanate, MGITC) molecules are sandwiched between a gold nanostar core and a thin silica shell. Such a SERS probe brings enhanced signal and low background fluorescence, shows good water-solubility and stability, and exhibits no sign of photobleaching. The aptamer labeled with the SERS probe is designed to hybridize with the cDNA on a gold film to form a rigid duplex DNA. In the presence of ATP, the interaction between ATP and the aptamer results in the dissociation of the duplex DNA structure and thereby removal of the SERS probe from the gold film, reducing the Raman signal. The response of the SERS biosensor varies linearly with the logarithmic ATP concentration up to 2.0 nM with a limit of detection of 12.4 pM. Our work has provided an effective method for detection of small molecules with SERS.     

Fluorescent detection of ATP based on signaling DNA aptamer attached silica nanoparticles

Novel methods for rapid, sensitive and low-cost biomolecule detection have attracted particular interest because of their wide use in medical diagnostics, food inspection and biomedical research applications. In this work, we report a simple and efficient silica nanoparticle (NP)-based fluorescent assay for ATP detection. It takes advantage of the washing and separation properties of NPs and the structure-switch property of DNA aptamers, resulting in fluorescence change of the supernatant in the presence of targets. A linear response for ATP detection was observed from 0 to 6?mM with a detection limit of ～34?μM. This detection strategy could be generalized to other aptamer-based detection systems.     

General colorimetric detection of proteins and small molecules based on cyclic enzymatic signal amplification and hairpin aptamer probe

In this work, we developed a simple and general method for highly sensitive detection of proteins and small molecules based on cyclic enzymatic signal amplification (CESA) and hairpin aptamer probe. Our detection system consists of a hairpin aptamer probe, a linker DNA, two sets of DNA-modified AuNPs, and nicking endonuclease (NEase). In the absence of a target, the hairpin aptamer probe and linker DNA can stably coexist in solution. Then, the linker DNA can assemble two sets of DNA-modified AuNPs, inducing the aggregation of AuNPs. However, in the presence of a target, the hairpin structure of aptamer probe is opened upon interaction with the target to form an aptamer probe-target complex. Then, the probe-target complex can hybridize to the linker DNA. Upon formation of the duplex, the NEase recognizes specific nucleotide sequence and cleaves the linker DNA into two fragments. After nicking, the released probe-target complex can hybridize with another intact linker DNA and the cycle starts anew. The cleaved fragments of linker DNA are not able to assemble two sets of DNA-modified AuNPs, thus a red color of separated AuNPs can be observed. Taking advantage of the AuNPs-based sensing technique, we are able to assay the target simply by UV-vis spectroscopy and even by the naked eye. Herein, we can detect the human thrombin with a detection limit of 50 pM and adenosine triphosphate (ATP) with a detection limit of 100 nM by the naked eye. This sensitivity is about 3 orders of magnitude higher than that of traditional AuNPs-based methods without amplification. In addition, this method is general since there is no requirement of the NEase recognition site in the aptamer sequence. Furthermore, we proved that the proposed method is capable of detecting the target in complicated biological samples.     

Electrogenerated chemiluminescence DNA biosensor based on hairpin DNA probe labeled with ruthenium complex

A highly selective electrogenerated chemiluminescence (ECL) biosensor for the detection of target single-strand DNA (ss-DNA) was developed using hairpin DNA as the recognition element and ruthenium complex as the signal-producing compound. The ECL-based DNA biosensor was fabricated by self-assembling the ECL probe of thiolated hairpin DNA tagged with ruthenium complex on the surface of a gold electrode. In the absence of target ss-DNA, the ECL probe immobilized on the surface of the electrode was in the folded configuration in which its termini were held in close proximity to the electrode, and thus a strong ECL signal could be generated. In the presence of target ss-DNA, a stem-loop of the ECL probe on the electrode was converted into a linear double-helix configuration due to hybridization, resulting in the tag moving away from the electrode surface, which in turn decreased the ECL signal. The ECL intensity of the DNA biosensor generated a "switch off" mode, which decreased with an increase of the concentration of target DNA, and a detection limit of 9 x 10(-11) M complementary target ss-DNA was achieved. Single mismatched target ss-DNA was effectively discriminated from complementary target ss-DNA. The effect of different loop lengths of the hairpin DNA on the selectivity of the ECL DNA biosensor has been investigated. This work demonstrated that the sensitivity and specificity of an ECL DNA biosensor could be greatly improved using a hairpin DNA species which has an appropriate stem and loop length as the recognition element.     

Impedimetric biosensor based on magnetic nanoparticles for electrochemical detection of dopamine

One of the difficulties which limit the use of electrochemical sensors for detection of dopamine is the interference from ascorbic acid. We have sought to address this problem through the synthesis and characterization of a suitable electrode material based on magnetic nanoparticles. The interference from the ascorbic acid was overcome by fabricating a negatively charged electrode surface using PEGylated arginine functionalized magnetic nanoparticles (PA-MNPs). The nanoparticles were characterized by various techniques viz., X-ray diffraction, FT-Infrared spectroscopy, transmission electron microscopy and vibrating sample magnetometer. The electrochemical behavior of the proposed sensor was investigated by cyclic voltammetry and the sensor showed high sensitivity and selectivity for dopamine. The response mechanism of the modified electrode is based on the interaction between the negatively charged electrode and the positively charged dopamine. Under optimized conditions, linear calibration plots were obtained for amperometric detection of dopamine (DA) over the concentration range of 1–9 mM dopamine, with a linear correlation coefficient of 0.9836, sensitivity of 121 μA/mM and a detection limit of 7.25 μM. Electrochemical impedance spectroscopy (EIS) has been used to study the interface properties of modified electrodes. The value of the polarization resistance (R_p) increases linearly with dopamine concentration in the range of 10 μM to 1 mM and the limit of detection (LOD) was calculated to be 14.1 μM. High sensitivity and selectivity, micromolar detection limit, high reproducibility, along with ease of preparation of the electrode surface make this system suitable for the determination of DA in pharmaceutical and clinical preparations.     

Electrochemical DNA biosensor based on conducting polyaniline nanotube array

Most of the recent developments in ultrasensitive detection of nucleic acid are based on the gold nanoparticles and carbon nanotubes as a medium of signal amplification. Here, we present an ultrasensitive electrochemical nucleic acid biosensor using the conducting polyaniline (PANI) nanotube array as the signal enhancement element. The PANI nanotube array of a highly organized structure was fabricated under a well-controlled nanoscale dimension on the graphite electrode using a thin nanoporous layer as a template, and 21-mer oligonucleotide probes were immobilized on these PANI nanotubes. In comparison with gold nanoparticle- or carbon nanotube-based DNA biosensors, our PANI nanotube array-based DNA biosensor could achieve similar sensitivity without catalytic enhancement, purification, or end-opening processing. The electrochemical results showed that the conducting PANI nanotube array had a signal enhancement capability, allowing the DNA biosensor to readily detect the target oligonucleotide at a concentration as low as 1.0 fM (approximately 300 zmol of target molecules). In addition, this biosensor demonstrated good capability of differentiating the perfect matched target oligonucleotide from one-nucleotide mismatched oligonucleotides even at a concentration of 37.59 fM. This detection specificity indicates that this biosensor could be applied to single-nucleotide polymorphism analysis and single-mutation detection.     

Electrochemical biosensor based on integrated assembly of dehydrogenase enzymes and gold nanoparticles

Development of a highly sensitive nanostructured electrochemical biosensor based on the integrated assembly of dehydrogenase enzymes and gold (Au) nanoparticle is described. The Au nanoparticles (AuNPs) have been self-assembled on a thiol-terminated, sol-gel-derived, 3-D, silicate network and enlarged by hydroxylamine seeding. The AuNPs on the silicate network efficiently catalyze the oxidation of NADH with a decrease in overpotential of approximately 915 mV in the absence of any redox mediator. The surface oxides of AuNP function as an excellent mediator, and a special inverted "V" shape voltammogram at less positive potential was observed for the oxidation of NADH. The AuNP self-assembled sol-gel network behaves like a nanoelectrode ensemble. The nanostructured electrode shows high sensitivity (0.056 +/- 0.001 nA/nM) toward NADH with an amperometric detection limit of 5 nM. The electrode displays excellent operational and storage stability. A novel methodology for the fabrication of a NADH-dependent dehydrogenase biosensor based on the integration of dehydrogenase enzyme and AuNPs with the silicate network is developed. The enzymatically generated NADH is, in turn, electrocatalytically detected by the AuNPs on the silicate network. The integrated assembly has been successfully used for the amperometric biosensing of lactate and ethanol at a potential of -5 mV. The biosensor is very stable and highly sensitive, and it has a fast response time. The excellent performance validates the integrated assembly as an attractive sensing element for the development of new dehydrogenase biosensors.     

One-step homogeneous protein detection based on aptamer probe and fluorescence cross-correlation spectroscopy

A new protein assay based on fluorescence cross-correlation spectroscopy (FCCS) and aptamer probe is developed. In this assay, two spectrally distinct fluorophores labeled aptamer probes are used to recognize and detect thrombin through a sandwich reaction. The sandwich complexes are diffused through a confocal detection volume. The cross-correlation signals can be observed only at the presence of the aptamer probes-protein sandwich complexes. Thrombin is selected as a target to validate the assay. The whole detection process can be completed within an hour with low-nanomolar sensitivity and high specificity. The novel aptamer-based FCCS detection offers a simple, rapid and sensitive method for protein analysis in a homogeneous format.     

Disposable biosensor based on Au nanoparticles-modified CdS nanorod arrays for detection cytochrome c

A novel disposable biosensor is developed based on gold nanoparticles modified CdS nanorod arrays. The ordered CdS nanorod arrays firstly have been synthesized by a simple hydrothermal method. Then, the CdS nanorod arrays are modified by gold nanoparticles, which are directly fabricated into an electrode for detection of cytochrome c (Cyc) in solution without any pretreatment. The modified CdS nanorod arrays biosensor with the immense surface area and high electrical conductivity shows a good sensitivity and linear range. This method may be used to construct other electrochemical biosensors using aligned nanorod/nanowire films.     

Immobilization of Prussian Blue nanoparticles onto thiol SAM modified Au electrodes for electroanalytical or biosensor applications

Poly(vinylpyrrolidone) (PVP)-protected Prussian Blue (PB) nanoparticles were prepared by simply mixing FeCI3 and K4Fe(CN)6 with absence or presence of HCI or/and KCI in water solution. The obtained PB nanoparticles were immobilized onto thiol self-assembled monolayer (SAM) modified Au electrodes. L-cysteine (Cys) and 1,8-octanedithiol (ODT) were compared as a bridge between the gold surface and the PB nanoparticles. The results show that PB prepared from the initial solution with KCI gives preferred electrochemical response and that Cys/Au shows improved immobilization effect of PB than ODT/Au. The obtained PB/Cys/Au electrodes exhibit electrocatalytic activity toward H2O2 reduction and DL-homocysteine (HCys) oxidation. Glucose oxidase (GOX) was immobilized onto PB modified electrode to explore the potentials for the design of oxidase-based biosensors. It is possible to anchor PB nanoparticles and develop their application on electroanalysis and biosensing.     

Carcinoembryonic antigen admittance biosensor based on Au and ZnO nanoparticles using FFT admittance voltammetry

In this work, a highly sensitive carcinoembryonic antigen fast Fourier transform admittance biosensor is introduced. The proposed biosensor is based on bilayer films of ZnO/Au nanoparticles as an immobilization matrix. These layers are prepared by self-assembly and deposition method on a gold electrode surface, respectively. Carcinoembryonic antibody (anti-CEA) was immobilized on gold nanoparticles and positively charged horseradish peroxidase (HRP) was used to block sites against nonspecific binding. The admittance biosensor was developed based on fast Fourier transform continuous square wave voltammetry, which produces a sensitive, fast (less than 20 s) and reliable response for determination of carcinoembryonic antigen. The technique was applied as a detector in a flow injection system. The admittances reduction current of the biosensor decreases linearly in two concentrations ranges of CEA from 0.1 to 70 ng/mL and from 70 to 200 ng/mL with a detection limit of 0.01 ng/mL in presence of 0.5 mM H(2)O(2) as an eluent solution.     

Detection of anticancer drug tamoxifen using biosensor based on polyaniline probe modified with horseradish peroxidase

Amperometric biosensor based on horseradish peroxidase immobilized via glutaraldehyde on the polyaniline modified platinum electrode shows evidenced promising characteristics in detecting anticancer drug tamoxifen. The sensor was fabricated simply by adsorbing horseradish peroxidase enzyme on the electrode surface for which Cyclic Voltammetry was used to monitor the electro-catalytic reduction of tamoxifen under diffusion-adsorption controlled conditions. Fourier Transform Infrared Spectroscopy, Cyclic Voltammetry and Electrochemical Impedance Spectroscopic techniques are used to characterize the electrochemical interfacial properties of surface modified electrodes. The first-hand effort on modified biosensor within Platinum/Polyaniline/Horseradish peroxidase biosensor system has demonstrated excellent electro-analytical properties with biosensor sensitivity of 1.6 μA ng mL^? 1. The optimum limit of detection and limit of quantification are 0.07 ng mL^? 1 and 0.29 ng mL^? 1 respectively for the determination of anticancer drug tamoxifen. It is felt that the present study will help in improving our knowledge of cost-effective quantitative determination of tamoxifen in metabolized biological fluids and other pharmaceutical formulations.     

Amperometric biosensor based on carbon nanotubes coated with polyaniline/dendrimer-encapsulated Pt nanoparticles for glucose detection

A novel amperometric glucose biosensor based on the nanocomposites of multi-wall carbon nanotubes (CNT) coated with polyaniline (PANI) and dendrimer-encapsulated Pt nanoparticles (Pt-DENs) is prepared. CNT coated with protonated PANI is in situ synthesized and Pt-DENs is absorbed on PANI/CNT composite surface by self-assembly method. Then Glucose oxidase (GOx) is crosslink-immobilizated onto Pt-DENs/PANI/CNT composite film. The results show that the fabricated GOx/Pt-DENs/PANI/CNT electrode exhibits excellent response performance to glucose, such as low detection limit (0.5 µM), wide linear range (1 µM–12 mM), short response time (about 5 s), high sensitivity (42.0 µA mM^? 1 cm^? 2) and stability (83% remains after 3 weeks).     

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.     

Nano‐biosensor based on reduced graphene oxide and gold nanoparticles,for detection of phenylketonuria‐associated DNA mutation

Phenylketonuria (PKU)‐associated DNA mutation in newborn children can be harmful to his health and early detection is the best way to inhibit consequences. A novel electrochemical nano‐biosensor was developed for PKU detection, based on signal amplification using nanomaterials, e.g. gold nanoparticles (AuNPs) decorated on the reduced graphene oxide sheet on the screen‐printed carbon electrode. The fabrication steps were checked by field emission scanning electron microscope imaging as well as cyclic voltammetry analysis. The specific alkanethiol single‐stranded DNA probes were attached by self‐assembly methodology on the AuNPs surface and Oracet blue was used as an intercalating electrochemical label. The results showed the detection limit of 21.3 fM and the dynamic range of 80–1200 fM. Moreover, the selectivity results represented a great specificity of the nano‐biosensor for its specific target DNA oligo versus other non‐specific sequences. The real sample simulation was performed successfully with almost no difference than a synthetic buffer solution environment.Inspec keywords: biosensors, nanosensors, nanoparticles, graphene compounds, gold, nanomedicine, DNA, molecular biophysics, biomedical equipment, electrochemical sensors, electrochemical electrodes, field emission scanning electron microscopy, voltammetry (chemical analysis), self‐assembly, biochemistryOther keywords: reduced graphene oxide, gold nanoparticles, phenylketonuria‐associated DNA mutation, newborn children, electrochemical nanobiosensor, signal amplification, nanomaterials, reduced graphene oxide sheet, screen‐printed carbon electrode, field emission scanning electron microscopy imaging, cyclic voltammetry, alkanethiol single‐stranded DNA probes, self‐assembly methodology, Oracet blue, intercalating electrochemical label, Au‐CO     

Visual cocaine detection with gold nanoparticles and rationally engineered aptamer structures

A novel bioassay strategy is designed to detect small-molecule targets such as cocaine, potassium, and adenosine, based on gold nanoparticles (AuNPs) and engineered DNA aptamers. In this design, an aptamer is engineered to be two pieces of random, coil-like single-stranded DNA, which reassembles into the intact aptamer tertiary structure in the presence of the specific target. AuNPs can effectively differentiate between these two states via their characteristic surface-plasmon resonance-based color change. Using this method, cocaine in the low-micromolar range is selectively detected within minutes. This strategy is also shown to be generic and applicable to the detection of several other small-molecule targets.     

DNA biosensor for the detection of hydrazines

A double-stranded (ds) DNA-coated carbon paste electrode is employed as a remarkably sensitive biosensor for the detection of hydrazine compounds. The sensor relies on monitoring changes in the intrinsic anodic response of the surface-confined DNA resulting from its interaction with hydrazine compounds and requires no label or indicator. Short reaction times (1-10 min) are sufficient for monitoring part-per-billion levels of different hydrazines. Applicability to untreated natural water samples is illustrated. The response mechanism is discussed, along with prospects of using DNA biosensors for quantitaing other important molecules and elucidating DNA interactions and damage.