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.     

Specific detection of Mycobacterium sp. genomic DNA using dual labeled gold nanoparticle based electrochemical biosensor

The present study was aimed at the development and evaluation of a DNA electrochemical biosensor for Mycobacterium sp. genomic DNA detection in a clinical specimen using a signal amplifier as dual-labeled AuNPs. The DNA electrochemical biosensors were fabricated using a sandwich detection strategy involving two kinds of DNA probes specific to Mycobacterium sp. genomic DNA. The probes of enzyme ALP and the detector probe both conjugated on the AuNPs and subsequently hybridized with target DNA immobilized in a SAM/ITO electrode followed by characterization with CV, EIS, and DPV analysis using the electroactive species para-nitrophenol generated by ALP through hydrolysis of para-nitrophenol phosphate. The effect of enhanced sensitivity was obtained due to the AuNPs carrying numerous ALPs per hybridization and a detection limit of 1.25 ng/ml genomic DNA was determined under optimized conditions. The dual-labeled AuNP-facilitated electrochemical sensor was also evaluated by clinical sputum samples, showing a higher sensitivity and specificity and the outcome was in agreement with the PCR analysis. In conclusion, the developed electrochemical sensor demonstrated unique sensitivity and specificity for both genomic DNA and sputum samples and can be employed as a regular diagnostics tool for Mycobacterium sp. monitoring in clinical samples.     

A simple and direct electrochemical detection of interferon-gamma using its RNA and DNA aptamers

Tuberculosis is the most frequent cause of infection-related death worldwide. We constructed a simple and direct electrochemical sensor to detect interferon (IFN)-gamma, a selective marker for tuberculosis pleurisy, using its RNA and DNA aptamers. IFN-gamma was detected by its 5'-thiol-modified aptamer probe immobilized on the gold electrode. Interaction between IFN-gamma and the aptamer was recorded using electrochemical impedance spectroscopy and quartz crystal microbalance (QCM) with high sensitivity. The RNA-aptamer-based sensor showed a low detection limit of 100 fM, and the DNA-aptamer-based sensor detected IFN-gamma to 1 pM in sodium phosphate buffer. With QCM analysis, the aptamer immobilized on the electrode and IFN-gamma bound to the aptamer probe was quantified. This QCM result shows that IFN-gamma exists in multimeric forms to interact with the aptamers, and the RNA aptamer prefers the high multimeric state of IFN-gamma. Such a preference may describe the low detection limit of the RNA aptamer shown by impedance analysis. In addition, IFN-gamma was detected to 10 pM by the DNA aptamer in fetal bovine serum, a mimicked biological system, which has similar components to pleural fluid.     

Sub-femtomolar electrochemical detection of DNA using surface circular strand-replacement polymerization and gold nanoparticle catalyzed silver deposition for signal amplification

A highly sensitive method was developed for detection of target DNA. This method combined circular strand-displacement polymerization (CSRP) with silver enhancement to achieve dual signal amplification. After molecular beacon (MB) hybridized with target DNA, the reporter gold nanoparticle (Au NPs) was attached to an electrode surface by hybridization between Au NP labeled primer and stem part of the MB to initiate a polymerization of DNA strand, which led to the release of target and another polymerization cycle. Thus the CSRP produced the multiplication of target-related reporter Au NPs on the surface. The Au NPs then catalyzed silver deposition for subsequent stripping analysis of silver. The dual signal amplification offered a dramatic enhancement of the stripping response. This signal could discriminate perfect matched target DNA from 1-base mismatch DNA. The dynamic range of the sequence-specific DNA detection was from 10(-16) to 10(-12)molL(-1) with a detection limit down to sub-femtomolar level. This proposed method exhibited an efficient amplification performance, and would open new opportunities for sensitive detection of other biorecognition events.     

Electrochemical synthesis of gold nanostructure modified electrode and its development in electrochemical DNA biosensor

In this article, gold nanostructure modified electrodes were achieved by a simple one-step electrodeposition method. The morphologies of modified electrodes could be easily controlled by changing the pH of HAuCl₄ solution. The novel nanoflower-like particles with the nanoplates as the building blocks could be interestingly obtained at pH 5.0. The gold nanoflower modified electrodes were then used for the fabrication of electrochemical DNA biosensor. The DNA biosensor fabrication process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the use of ferricyanide as an electrochemical redox indicator. The DNA immobilization and hybridization on gold nanoflower modified electrode was studied with the use of [Ru(NH₃)₆]³⁺ as a hybridization indicator. The electrochemical DNA biosensor shows a good selectivity and sensitivity toward the detection of target DNA. A detection limit of 1 pM toward target DNA could be obtained.     

A novel electrochemical sensor surface for the detection of hydrogen peroxide using cyclic bisureas/gold nanoparticle composite

A novel electrochemical sensor surface with enhanced sensitivity for the detection of hydrogen peroxide has been developed based on the layer-by-layer assembly of mercapto propionic acid (MPA), cystine-based polymethylene-bridged cyclic bisureas (CBU)/gold nanoparticle (AuNP) and horseradish peroxidase (HRP) on gold electrode. Possibility of a large number of hydrogen bonds, allowed by the chemical and sterical structure of the CBU ensures the proper immobilization of the enzyme in favorable orientation and retention of enzymatic activity. Efficient electron tunneling property of AuNP together with its electrocatalytic activity leads to higher sensitivity in the detection of H(2)O(2). In cyclic voltammetry measurements a cathodic current due to direct electron transfer of HRP is observed which, indicates excellent electrocatalytic activity of the sensor surface. The biosensor surface modified with gold nanoparticle and CBU showed a lower detection limit of 50 nM for hydrogen peroxide. Chronoamperometry is performed at -0.3 V and Michaelis-Menten constant K(M)(app) value is estimated to be 4.5 μM. The newly developed sensor surface showed very high stability, reproducibility and high sensitivity.     

Detection of parathyroid hormone using an electrochemical impedance biosensor based on PAMAM dendrimers

This paper presents a novel hormone‐based impedimetric biosensor to determine parathyroid hormone (PTH) level in serum for diagnosis and monitoring treatment of hyperparathyroidism, hypoparathyroidism and thyroid cancer. The interaction between PTH and the biosensor was investigated by an electrochemical method. The biosensor was based on the gold electrode modified by 12‐mercapto dodecanoic (12MDDA). Antiparathyroid hormone (anti‐PTH) was covalently immobilized on to poly amidoamine dendrimer (PAMAM) which was bound to a 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide/N‐hydroxysuccinimide (EDC/NHS) couple, self‐assembled monolayer structure from one of the other NH₂ sites. The immobilization of anti‐PTH was monitored by electrochemical impedance spectroscopy, cyclic voltammetry and scanning electron microscope techniques. After the optimization studies of immobilization materials such as 12MDDA, EDC–NHS, PAMAM, and glutaraldehyde, the performance of the biosensor was investigated in terms of linearity, sensitivity, repeatability, and reproducibility. PTH was detected within a linear range of 10–60 fg/mL. Finally the described biosensor was used to monitor PTH levels in artificial serum samples. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:815–822, 2015     

Trace detection of picloram using an electrochemical immunosensor based on three-dimensional gold nanoclusters

Picloram, a herbicide widely used for broadleaf weed control, is persistent and mobile in soil and water with adverse health and environmental effects. It is important to develop a sensitive method for accurate detection of trace picloram in the environment. In this article, a type of ordered three-dimensional (3D) gold (Au) nanoclusters obtained by two-step electrodeposition using the spatial obstruction/direction of the polycarbonate membrane is reported. Bovine serum albumin (BSA)-picloram was immobilized on the 3D Au nanoclusters by self-assembly, and then competitive immunoreaction with picloram antibody and target picloram was executed. The horseradish peroxidase (HRP)-labeled secondary antibody was applied for enzyme-amplified amperometric measurement. The electrodeposited Au nanoclusters built direct electrical contact and immobilization interface with protein molecules without postmodification and positioning. Under the optimal conditions, the linear range for picloram determination was 0.001-10 μg/ml with a correlation coefficient of 0.996. The detection and quantification limits were 5.0 × 10⁻⁴ and 0.0021 μg/ml, respectively. Picloram concentrations in peach and excess sludge supernatant extracts were tested by the proposed immunosensor, which exhibited good precision, sensitivity, selectivity, and storage stability.     

Green synthesis of conductive polyaniline by Trametes versicolor laccase using a DNA template

The green synthesis of highly conductive polyaniline by using two biological macromolecules, i.e laccase as biocatalyst, and DNA as template/dopant, was achieved in this work. Trametes versicolor laccase B (TvB) was found effective in oxidizing both aniline and its less toxic/mutagenic dimer N‐phenyl‐p‐phenylenediamine (DANI) to conductive polyaniline. Reaction conditions for synthesis of conductive polyanilines were set‐up, and structural and electrochemical properties of the two polymers were extensively investigated. When the less toxic aniline dimer was used as substrate, the polymerization reaction was faster and gave less‐branched polymer. DNA was proven to work as hard template for both enzymatically synthesized polymers, conferring them a semi‐ordered morphology. Moreover, DNA also acts as dopant leading to polymers with extraordinary conductive properties (～6 S/cm). It can be envisaged that polymer properties are magnified by the concomitant action of DNA as template and dopant. Herein, the developed combination of laccase and DNA represents a breakthrough in the green synthesis of conductive materials.     

The detection of a transgenic soybean biochip using gold label silver stain technology

A method for the rapid detection of transgenic soybean crops based on a combination of gene chip and "gold label silver stain" (GLSS) technologies has been established. To ensure the specificity of this method, the CaMV35S promoter and Nos terminator were selected as probes because they are both exogenous genes that are specific to transgenic soybean plants. The addition of biotin-modified dUTPs to a polymerase chain reaction (PCR) system can produce amplified nucleic acid segments containing biotin. These labeled PCR products then hybridize with specific probes on the chip and are subsequently bound by streptavidin-modified gold nanoparticles (GNPs). Due to the catalytic nature of the GNPs, silver staining can be used to visualize the hybridized probes, which appear as signals in varying shades of gray. The intensity value of the gray signals can be obtained using a general scanner. Silver staining for 10 min was determined to produce the optimal signal-to-noise ratio. In addition, this method was shown to be highly specific and had a detection sensitivity of 288.57 pg/μL.     

Preparation of novel mercury-doped silver nanoparticles film glassy carbon electrode and its application for electrochemical biosensor

A novel mercury-doped silver nanoparticles film glassy carbon (Ag/MFGC) electrode was prepared in this study. Electrochemical behaviors of cysteine on the Ag/MFGC electrode were investigated by electrochemical impedance spectroscopy and cyclic voltammetry (CV). The results indicated that cysteine could be strongly adsorbed on the surface of the Ag/MFGC electrode to form a thin layer. The doped electrode could catalyze the electrode reaction process of cysteine, and the cysteine displayed a pair of well-defined and nearly reversible CV peaks at the electrode in an acetate buffer solution (pH 5.0). The Ag/MFGC electrode was used for determination of cysteine by differential pulse voltammetry. The linear range was between 4.0x10(-7) and 1.3x10(-5) mol/L, with a detection limit of 1.0x10(-7) mol/L and a signal-to-noise ratio of 3. The relative standard deviation was 2.4% for seven successive determinations of 1.0x10(-5) mol/L cysteine. The determinations of cysteine in synthetic samples and urinal samples were carried out and satisfactory results were obtained. Amperometric application of the Ag/MFGC electrode as biosensors is proposed.     

Simultaneous detection of two tumor markers using silver and gold nanoparticles decorated carbon nanospheres as labels

In this work, a multiplexed electrochemical immunosensor was developed for sensitive detection of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) using silver nanoparticles (Ag NPs) or gold nanoparticles (Au NPs) coated-carbon nanospheres (CNSs) as labels. CNSs were employed as the carrier for the immobilization of nanoparticles (Ag NPs or Au NPs), thionine (Thi), and secondary antibodies (Ab₂) due to their good monodispersity and uniform structure. Au NPs reduced graphene oxide (rGO) nanocomposites were used as sensing substrate for assembling two primary antibodies (Ab₁). In the presence of target proteins, two labels were attached onto the surface of the rGO/Au NPs nanocomposites via a sandwich immunoreaction. Two distinguishable peaks, one at +0.16 V (corresponding to Ag NPs) and another at −0.33 V (corresponding to Thi), were obtained in differential pulse voltammetry (DPV). The peak difference was approximately 490 mV, indicating that CEA and AFP can be simultaneously detected in a single run. Under optimal conditions, the peak currents were linearly related to the concentrations of CEA or AFP in the range of 0.01–80 ng ml⁻¹. The detection limits of CEA and AFP were 2.8 and 3.5 pg ml⁻¹, respectively (at a signal-to-noise ratio of 3). Moreover, when the immunosensor was applied to serum samples, the results obtained were in agreement with those of the reference method, indicating that the immunosensor would be promising in the application of clinical diagnosis and screening of biomarkers.     

Time-course correlation of biofilm properties and electrochemical performance in single-chamber microbial fuel cells

The relationship between anode microbial characteristics and electrochemical parameters in microbial fuel cells (MFCs) was analyzed by time-course sampling of parallel single-bottle MFCs operated under identical conditions. While voltage stabilized within 4 days, anode biofilms continued growing during the six-week operation. Viable cell density increased asymptotically, but membrane-compromised cells accumulated steadily from only 9% of total cells on day 3 to 52% at 6 weeks. Electrochemical performance followed the viable cell trend, with a positive correlation for power density and an inverse correlation for anode charge transfer resistance. The biofilm architecture shifted from rod-shaped, dispersed cells to more filamentous structures, with the continuous detection of Geobacter sulfurreducens-like 16S rRNA fragments throughout operation and the emergence of a community member related to a known phenazine-producing Pseudomonas species. A drop in cathode open circuit potential between weeks two and three suggested that uncontrolled biofilm growth on the cathode deleteriously affects system performance.     

Application of electrochemical impedance spectroscopy for monitoring allergen-antibody reactions using gold nanoparticle-based biomolecular immobilization method

Gold nanoparticles were used to enhance the immobilization amount and retain the immunoactivity of recombinant dust mite allergen Der f2 immobilized on a glassy carbon electrode (GCE). The interaction between allergen and antibody was studied by electrochemical impedance spectroscopy (EIS). Self-assembled Au colloid layer (?=16nm) deposited on (3-mercaptopropyl)trimethoxysilane (MPTS)-modified GCE offered a basis to control the immobilization of allergen Der f2. The impedance measurements were based on the charge transfer kinetics of the [Fe(CN)(6)](3-/4-) redox pair, compared with bare GCE, the immobilization of allergen Der f2 and the allergen-antibody interaction that occurred on the electrode surface altered the interfacial electron transfer resistance and thereby slowed down the charge transfer kinetics by reducing the active area of the electrode or by preventing the redox species in electrolyte solution from approaching the electrode. The interactions of allergen with various concentrations of monoclonal antibody were also monitored through the change of impedance response. The results showed that the electron transfer resistance increased with increasing concentrations of monoclonal antibody.     

电化学生物传感器快速检测DNA研究进展

本简要地介绍了DNA电化学生物传感器研究的最新进展,重点讨论了改善生物传感器选择性和灵敏度的技术和方法。     

Highly sensitive biosensor based on aptamer and hybridization chain reaction for detection of cadmium ions

In this work, a highly sensitive biosensor for detecting cadmium ions (Cd²⁺) was developed based on a Cd²⁺-specific DNA aptamer and a hybridization chain reaction (HCR). The Cd²⁺ aptamer (named S0) was used to recognize Cd²⁺ and trigger the HCR. Without Cd²⁺, S0 initiated the HCR to form long nicked dsDNA structures to quench the fluorescence. Then, Cd²⁺ could bind with S0 to block HCR to recover fluorescence. This biosensor had high sensitivity with a detection limit of 0.36 nM and a linear range from 0 to 10 nM. Moreover, it showed a satisfactory selectivity and recovery rates.     

Continuous flow system for biofilm formation using controlled concentrations of Pseudomonas putida from chicken carcass and coupled to electrochemical impedance detection

Abstract

Most studies dealing with monitoring the dynamics of biofilm formation use microbial suspensions at high concentrations. These conditions do not always represent food or water distribution systems. A continuous flow system capable of controlling the concentration of the microbial suspension stream from 10⁴ to 10⁶ CFU ml^?1 is reported. Pseudomonas putida biofilms formed using 100-fold, 1,000-fold or 10,000-fold diluted bacterial suspensions were monitored in-line by electrochemical impedance spectroscopy (EIS) and total plate counts. Randles equivalent circuit model and a modified Randles model with biofilm elements were used to fit the EIS data. In Randles equivalent circuit, the charge transfer resistance decreased as the biofilm formed. The log colony counts of the biofilm correlated to the charge transfer resistance. In the biofilm model, the biofilm resistance and the double layer capacitance decreased as the biofilm formed. The log colony counts of the biofilm correlated to the biofilm resistance.     

Electrochemical measurement of DNA hybridization using nanosilver as label and horseradish peroxidase as enhancer

A simple and sensitive electrochemical DNA biosensor based on in situ DNA amplification with nanosilver as label and horseradish peroxide (HRP) as enhancer has been designed. The thiolated oligomer single-stranded DNA (ssDNA) was initially directly immobilized on a gold electrode, and quartz crystal microbalance (QCM) gave the specific amount of ssDNA adsorption of 6.3 ± 0.1 ng/cm². With a competitive format, hybridization reaction was carried out via immersing the DNA biosensor into a stirred hybridization solution containing different concentrations of the complementary ssDNA and constant concentration of nanosilver-labeled ssDNA, and then further binding with HRP. The adsorbed HRP amount on the probe surface decreased with the increment of the target ssDNA in the sample. The hybridization events were monitored by using differential pulse voltammetry (DPV) with the adsorbed HRP toward the reduction of H₂O₂. The reduction current from the enzyme-generated product was related to the number of target ssDNA molecules in the sample. A detection of 15 pmol/L for target ssDNA was obtained with the electrochemical DNA biosensor. Additionally, the developed approach can effectively discriminate complementary from non-complementary DNA sequence, suggesting that the similar enzyme-labeled DNA assay method hold great promises for sensitive electrochemical biosensor applications.     

A nanoparticle label/immunochromatographic electrochemical biosensor for rapid and sensitive detection of prostate-specific antigen

We present a nanoparticle (NP) label/immunochromatographic electrochemical biosensor (IEB) for rapid and sensitive detection of prostate-specific antigen (PSA) in human serum. This IEB integrates the immunochromatographic strip with the electrochemical detector for transducing quantitative signals. The NP label, made of CdSe@ZnS, serves as a signal-amplifier vehicle. A sandwich immunoreaction was performed on the immunochromatographic strip. The captured NP labels in the test zone were determined by highly sensitive stripping voltammetric measurement of the dissolved metallic component (cadmium) with a disposable-screen-printed electrode, which is embedded underneath the membrane of the test zone. Several experimental parameters (e.g., immunoreaction time, the amount of anti-PSA-NP conjugations applied) and electrochemical detection conditions (e.g., preconcentration potential and time) were optimized using this biosensor for PSA detection. The analytical performance of this biosensor was evaluated with serum PSA samples according to the “figure-of-merits” (e.g., dynamic range, reproducibility, and detection limit). The results were validated with enzyme-linked immunosorbent assay (ELISA) and showed high consistency. It is found that this biosensor is very sensitive with the detection limit of 0.02 ng mL⁻¹ PSA and is quite reproducible (with a relative standard deviation (R.S.D.) of 6.4%). This method is rapid, clinically practical, and less expensive than other diagnostic tools for PSA; therefore, this IEB coupled with a portable electrochemical analyzer shows great promise for simple, sensitive, quantitative point-of-care testing of disease-related protein biomarkers.