Nanocomposite of electrochemically reduced graphene oxide and gold nanourchins for electrochemical DNA detection

A nanocomposite of graphene oxide and gold nanourchins has been used here to modify the surface of a screen‐printed carbon electrode to enhance the sensitivity of the electrochemical DNA detection system. A specific single‐stranded DNA probe was designed based on the target DNA sequence and was thiolated to be self‐assembled on the surface of the gold nanourchins placed on the modified electrode. Doxorubicin was used as an electrochemical label to detect the DNA hybridisation using differential pulse voltammetry (DPV). The assembling process was confirmed using scanning electron microscopy (SEM) imaging, cyclic voltammetry (CV), and the EIS method. The high sensitivity of the proposed system led to a low detection limit of 0.16 fM and a wide linear range from 0.5 to 950.0 fM. The specificity of the DNA hybridisation and the signalling molecule (haematoxylin) caused very high selectivity towards the target DNA than other non‐specific sequences.     

Magneto-controlled graphene immunosensing platform for simultaneous multiplexed electrochemical immunoassay using distinguishable signal tags

A novel flow-through multiplexed immunoassay protocol for simultaneous electrochemical determination of carcinoembryonic (CEA) and alpha-fetoprotein (AFP) in biological fluids was designed using biofunctionalized magnetic graphene nanosheets (MGO) as immunosensing probes and multifunctional nanogold hollow microspheres (GHS) as distinguishable signal tags. The probes were fabricated by means of co-immobilization of primary anti-CEA (Ab(1)) and anti-AFP (Ab(2)) antibodies on the Fe(3)O(4) nanoparticle-coated graphene nanosheets (MGO-Ab(1,2)). The reverse-micelle method was used for the synthesis of distinguishable signal tags by encapsulation of horseradish peroxide (HRP)-thionine and HRP-ferrocene into nanogold hollow microspheres, respectively, which were utilized as labels of the corresponding GHS-Ab(1) and GHS-Ab(2). A sandwich-type immunoassay format was employed for the online detection of CEA and AFP by coupling a flow-through detection cell with an external magnet. The assay was based on the catalytic reduction of H(2)O(2) at the various peak potentials in the presence of the corresponding mediators. Experimental results revealed that the multiplexed electrochemical immunoassay enabled the simultaneous monitoring of AFP and CEA in a single run with wide working ranges of 0.01-200 ng mL(-1) for AFP and 0.01-80 ng mL(-1) for CEA. The detection limits (LODs) for both analytes at 1.0 pg mL(-1) (at 3s(B)) were very low. No obvious nonspecific adsorption and cross-talk were observed during a series of analyses to detect target analytes. Intraassay and interassay coefficients of variation were <10%. Importantly, the methodology was evaluated for the analysis of clinical serum specimens, receiving a good correlation between the flow-through multiplexed electrochemical immunoassay and an electrochemiluminescence method as a reference.     

A reduced graphene oxide based electrochemical biosensor for tyrosine detection

In this paper, a 'green' and safe hydrothermal method has been used to reduce graphene oxide and produce hemin modified graphene nanosheet (HGN) based electrochemical biosensors for the determination of l-tyrosine levels. The as-fabricated HGN biosensors were characterized by UV-visible absorption spectra, fluorescence spectra, Fourier transform infrared spectroscopy (FTIR) spectra and thermogravimetric analysis (TGA). The experimental results indicated that hemin was successfully immobilized on the reduced graphene oxide nanosheet (rGO) through π-π interaction. TEM images and EDX results further confirmed the attachment of hemin on the rGO nanosheet. Cyclic voltammetry tests were carried out for the bare glass carbon electrode (GCE), the rGO electrode (rGO/GCE), and the hemin-rGO electrode (HGN/GCE). The HGN/GCE based biosensor exhibits a tyrosine detection linear range from 5?×?10(-7)?M to 2?×?10(-5)?M with a detection limitation of 7.5?×?10(-8)?M at a signal-to-noise ratio of 3. The sensitivity of this biosensor is 133 times higher than that of the bare GCE. In comparison with other works, electroactive biosensors are easily fabricated, easily controlled and cost-effective. Moreover, the hemin-rGO based biosensors demonstrate higher stability, a broader detection linear range and better detection sensitivity. Study of the oxidation scheme reveals that the rGO enhances the electron transfer between the electrode and the hemin, and the existence of hemin groups effectively electrocatalyzes the oxidation of tyrosine. This study contributes to a widespread clinical application of nanomaterial based biosensor devices with a broader detection linear range, improved stability, enhanced sensitivity and reduced costs.     

Gold nanoparticles decorated reduced graphene oxide nanolabel for voltammetric immunosensing

This study describes the development and testing of a simple and novel enzyme‐free nanolabel for the detection and signal amplification in a sandwich immunoassay. Gold nanoparticles decorated reduced graphene oxide (rGOAu) was used as the nanolabel for the quantitative detection of human immunoglobulin G (HIgG). The rGOAu nanolabel was synthesised by one pot chemical reduction of graphene oxide and chloroauric acid using sodium borohydride. The pseudo‐peroxidase behaviour of rGOAu makes the nanolabel unique from other existing labels. The immunosensing platform was fabricated using self‐assembled monolayers of 11‐mercaptoundecanoic acid (11‐MUDA) on a gold disc electrode. The covalent immobilisation of antibody was achieved through the bonding of the carboxyl group of 11‐MUDA and the amino group of the antibody using chemical linkers [1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide] and N ‐hydroxysuccinimide. The fabricated immunosensor exhibited a linear range that included HIgG concentrations of 62.5–500 ng ml⁻¹. The sensor was also used for the testing of HIgG in the blood sample.Inspec keywords: proteins, nanomedicine, reduction (chemical), chemical sensors, nanofabrication, electrochemical sensors, voltammetry (chemical analysis), gold, oxidation, self‐assembly, monolayers, molecular biophysics, biochemistry, biosensors, nanoparticles, nanosensors, blood, grapheneOther keywords: gold nanoparticles, voltammetric immunosensing, enzyme‐free nanolabel, signal amplification, sandwich immunoassay, human immunoglobulin G, rGOAu nanolabel, chloroauric acid, sodium borohydride, 11‐mercaptoundecanoic acid, 11‐MUDA, gold disc electrode, chemical linkers, 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide], HIgG concentrations, reduced graphene oxide nanolabel, quantitative HIgG detection, one pot chemical reduction, covalent antibody immobilisation, carboxyl group bonding, pseudo‐peroxidase behaviour, self‐assembled monolayers, N‐hydroxysuccinimide, immunosensor, blood sample, Au‐CO     

Triple signal amplification of graphene film, polybead carried gold nanoparticles as tracing tag and silver deposition for ultrasensitive electrochemical immunosensing

A triple signal amplification strategy was designed for ultrasensitive immunosensing of cancer biomarker. This strategy was achieved using graphene to modify immunosensor surface for accelerating electron transfer, poly(styrene-co-acrylic acid) microbead (PSA) carried gold nanoparticles (AuNPs) as tracing tag to label signal antibody (Ab(2)) and AuNPs induced silver deposition for anodic stripping analysis. The immunosensor was constructed by covalently immobilizing capture antibody on chitosan/electrochemically reduced graphene oxide film modified glass carbon electrode. The in situ synthesis of AuNPs led to the loading of numerous AuNPs on PSA surface and convenient labeling of the tag to Ab(2). With a sandwich-type immunoreaction, the AuNPs/PSA labeled Ab(2) was captured on the surface of an immunosensor to further induce a silver deposition process. The electrochemical stripping signal of the deposited silver nanoparticles in KCl was used to monitor the immunoreaction. The triple signal amplification greatly enhanced the sensitivity for biomarker detection. The proposed method could detect carcinoembryonic antigen with a linear range of 0.5 pg mL(-1) to 0.5 ng mL(-1) and a detection limit down to 0.12 pg mL(-1). The immunosensor exhibited good stability and acceptable reproducibility and accuracy, indicating potential applications in clinical diagnostics.     

Immunoassays with protein misfolding cycle amplification: a platform for ultrasensitive detection of antigen

Protein misfolding cycle amplification (PMCA), a novel technology on amplifying cyclically misfolded proteins in vitro, is conceptually analogous to DNA amplification by polymerase chain reaction (PCR) and has tremendous implications for the researches and diagnosis. Here we first introduce the protein amplification technology into the classic immunoassay and develop a PMCA-based immunoassay (immuno-PMCA) for highly sensitive detection of antigen. This method takes advantage of sandwich binding of two affinity aptamers for increased specificity, magnetic nanoparticles for fast magnetic separation, PMCA for signal amplification, and conjugated polyelectrolytes for visual detection, allowing the detection limit of antigen by colorimetry down to femtomolar level with a wide linear range from 10 to 10(4) fM. More importantly, no specialized facilities or enzymes are needed either in the amplification reaction or the evaluation of results, which indicates its great potential application in immunological research and clinical diagnostics.     

An ultrasensitive electrochemical immunosensor for hepatitis C antibodies based on one-step-eletrosynthetized polypyrrole–graphene nanocomposite

Journal of Materials Science - An ultrasensitive label-free electrochemical immunosensor was developed for hepatitis C antibodies (anti-HCV). Worldwide, it is estimated 71 million people have HCV...     

A platform for electrochemical sensing of biomolecules based on Europia/reduced graphene oxide nanocomposite

This study aimed at preparing and evaluating the europium oxide–reduced graphene oxide (rGO) composites. Inorganic nanoparticles anchored onto rGO sheets through a facile sonochemical method. The resultant products were characterized by FT-IR, XRD, SEM. Their activity in biomolecules’ analysis were examined by cyclic voltammetry. The rectified electrodes revealed an incredibly electroactive manner. The obtained progress provided excellent materials for scrutiny of biomolecules. The linear relationship was used in the region of 100–1500 µM ascorbic acid (AA), 50–600 µM dopamine (DA), and 10–700 µM uric acid (UA), between current intensities and concentrations. The detection restrictions (LOD) (S/N?=?3) decreased to 8 µM, 1.1 µM and 0.085 µM for AA, DA and UA respectively by differential pulse voltammetry (DPV).     

Particulate platform for bioluminescent immunosensing

The present study examines pyruvate kinase-conjugated antibodies for potential use in ELISA applications. The conjugates had an acceptable stability, and the coupling inflicted only minor impairment on the kinase activity. To mimic the setup of an immunoassay under development, a test antigen (BSA) was attached to polystyrene nanoparticles. This arrangement was found to be suitable as solid support for presentation of antigens in sensitive bioluminescence assays. The nanoparticles were well characterized in terms of protein surface load and were used to establish the number of conjugate complexes needed to generate a detectable signal. Under the biochemical conditions employed here, the detection limit of the pyruvate kinase conjugate lies in the femtomole range.     

Toward the early evaluation of therapeutic effects: an electrochemical platform for ultrasensitive detection of apoptotic cells

The ability for early evaluation of therapeutic effects is a significant challenge in leukemia research. To address this challenge, we developed a novel electrochemical platform for ultrasensitive and selective detection of apoptotic cells in response to therapy. In order to construct the platform, a novel three-dimensional (3-D) architecture was initially fabricated after combining nitrogen-doped carbon nanotubes and gold nanoparticles via a layer-by-layer method. The formed architecture provided an effective matrix for annexin V with high stability and bioactivity to enhance sensitivity. On the basis of the specific recognition between annexin V and phosphatidylserine on the apoptotic cell membrane, the annexin V/3-D architecture interface showed a predominant capability for apoptotic cell capture. Moreover, a lectin-based nanoprobe was designed by noncovalent assembly of concanavalin A on CdTe quantum dots (QDs)-labeled silica nanospheres with poly(allylamine hydrochloride) as a linker. This nanoprobe incorporated both the specific carbohydrate recognition and the multilabeled QDs-based signal amplification. By coupling with the QDs-based nanoprobe and electrochemical stripping analysis, the proposed sandwich-type cytosensor showed an excellent analytical performance for the ultrasensitive detection of apoptotic cells (as low as 48 cells), revealing great potential toward the early evaluation of therapeutic effects.     

Heterogeneous immunosensing using antigen and antibody monolayers on gold surfaces with electrochemical and scanning probe detection

We report the use of antibody and antigen monolayer immunosurfaces as detection elements in a competitive heterogeneous immunoassay employing either electrochemical or scanning probe detection. Antibody or antigen monolayers were prepared by covalent attachment of the desired immunoreagent to a two-component self-assembled monolayer via amide linkages. More specifically, mixed monolayers of a carboxylic acid-terminated thiol (thioctic acid) and a methyl-terminated thiol (butanethiol) were used to control the surface epitope density. The microscopic structure of the resulting antibody and antigen arrays was characterized by AFM (atomic force microscopy). Individual, surface-confined rabbit IgG antibodies could be directly imaged in contact mode. The average height of the capture antibodies was found to be 7.1 nm; the average antibody diameter, after correcting for tip convolution effects, was determined to be between 7 and 10 nm. The surface epitope density could be varied over approximately 2 orders of magnitude by changing the composition of the mixed monolayer. AFM was also used to characterize the antibody-antigen binding characteristics of these immunosurfaces, and an average binding efficiency of 22.8% was measured for rabbit IgG antibody arrays. In the second part of this study, the electrochemical detection scheme originally developed by Heineman and co-workers was adapted to our system. A calibration data set was measured, and the linear least-squares correlation coefficient (R2) was found to be 0.993. Finally, the electrochemical and scanning probe detection modes were directly compared. We find an excellent correlation between the surface density of antibody-antigen complexes measured by AFM and the electrochemical response of the same immunosurfaces.     

Application of electrochemically reduced graphene oxide on screen-printed ion-selective electrode

In this study, a novel disposable all-solid-state ion-selective electrode using graphene as the ion-to-electron transducer was developed. The graphene film was prepared on screen-printed electrode directly from the graphene oxide dispersion by a one-step electrodeposition technique. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to demonstrate the large double layer capacitance and fast charge transfer of the graphene film modified electrode. On the basis of these excellent properties, an all-solid-state calcium ion-selective electrode as the model was constructed using the calcium ion-selective membrane and graphene film modified electrode. The mechanism about the graphene promoting the ion-to-electron transformation was investigated in detail. The disposable electrode exhibited a Nernstian slope (29.1 mV/decade), low detection limit (10(-5.8) M), and fast response time (less than 10 s). With the high hydrophobic character of graphene materials, no water film was formed between the ion-selective membrane and the underlying graphene layer. Further studies revealed that the developed electrode was insensitive to light, oxygen, and redox species. The use of the disposable electrode for real sample analysis obtained satisfactory results, which made it a promising alternative in routine sensing applications.     

Large-area nanopatterned graphene for ultrasensitive gas sensing

Chemical vapor deposited （CVD） graphene is nanopatterned using a spherical block copolymer etch mask. The use of spherical rather than cylindrical block copolymers allows homogeneous patterning of cm-scale areas without any substrate surface treatment. Raman spectroscopy was used to study the con- trolled generation of point defects in the graphene lattice with increasing etching time, confirming that alongside the nanomesh patterning, the nanopatterned CVD graphene presents a high defect density between the mesh holes. The nanopatterned samples showed sensitivities for NO2 of more than one order of magnitude higher than for non-patterned graphene. NO2 concentrations as low as 300 ppt were detected with an ultimate detection limit of tens of ppt. This is the smallest value reported so far for non-UV illuminated graphene chemiresistive NO2 gas sensors. The dramatic improvement in the gas sensitivity is believed to be due to the high adsorption site density, thanks to the combination of edge sites and point defect sites. This work opens the possibility of large area fabrication of nanopatterned graphene with extremely high densities of adsorption sites for sensing applications.     

Electroless deposition of Au nanoparticles on reduced graphene oxide/polyimide film for electrochemical detection of hydroquinone and catechol

An electrochemical sensor for determination of hydroquinone (HQ) and catechol (CC) was developed using Au nanoparticles (AuNPs) fabricated on reduced graphene oxide/polyimide (PI/RGO) film by electroless deposition. The electrochemical behaviors of HQ and CC at PI/RGO-AuNPs electrode were investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Under the optimized condition, the current responses at PI/RGO-AuNPs electrode were linear over ranges from 1 to 654 mol/L for HQ and from 2 to 1289 mol/L for CC, with the detection limits of 0.09 and 0.2 mol/L, respectively. The proposed electrode exhibited good reproducibility, stability and selectivity. In addition, the proposed electrode was successfully applied in the determination of HQ and CC in tap water and the Yellow River samples.     

An electrochemical route to graphene oxide

Large-scale graphene oxide (GO) with adjustable resistivity was synthesized from graphite via an electrochemical method using KCl solution as an effective electrolyte. During the exfoliation process, electrostatic force intercalates chloride ions between the expanded graphite layers on the anode. These chloride ions form small gas bubbles between the graphite layers in the electrochemical reaction. It is believed that the gas bubbles expand the gap between graphite sheets and produce a separating force between adjacent graphene layers. This separating force overcomes the Van der Waals force between adjacent sheets and exfoliates graphene layers from the starting graphite. Because the graphene is electrochemically oxidized by chorine during the exfoliation, the exfoliated GO sheets are hydrophilic and easily dispersed in the electrolyte solution. The GO solution prepared by the electrochemical exfoliation can be simply sprayed or spin-coated onto any substrate for device applications. The measured average thicknesses of a monolayer, bilayer, and trilayer exfoliated GO on SiO2 substrate were 1.9, 2.8, and 3.9 nm, respectively. It was observed that the measured resistance of the exfoliated GO sheets increases due to electrochemical oxidation in the solution. This electrochemical approach offers a low-cost and efficient route to the fabrication of graphene based devices.     

Self-assembly of quantum dots and carbon nanotubes for ultrasensitive DNA and antigen detection

A highly selective, ultrasensitive, fluorescence detection method for DNA and antigen based on self-assembly of multiwalled carbon nanotubes (CNTs) and CdSe quantum dots (QDs) via oligonucleotide hybridization is reported. Mercaptoalkyloligonucleotide molecules bind to the quantum dots, while amineoalkyloligonucleotides bind to CNTs with -COCl surface groups. QDs and CNTs further assemble into nanohybrids through DNA hybridization in the presence of target complementary oligonucleotides. The method is achieved with good repeatability with the detection limit of 0.2 pM DNA molecules and 0.01 nM antigen molecules. This novel detection system can also be used for multicomponent detection and antigen-antibody immunoreaction. The novel system has great potential in applications such as ultrasensitive pathogen DNA or antigen or antibody detection, molecular imaging, and photoelectrical biosensors.     

Optimizing label-free DNA electrical detection on graphene platform

The anodized epitaxial graphene (EG) electrode demonstrates a high level of performance for electrochemical impedance as well as differential pulse voltammetry detection of immobilized DNA and free DNA, respectively, at solid-liquid interfaces. On the anodized EG surface, because of the presence of oxygen functionalities as well as π conjugated domains, the anchoring of the DNA probe can be achieved by either covalent grafting or noncovalent π-π stacking readily. The effect of different binding modes on the sensitivity of the impedimetric sensing was investigated. Equivalent circuit modeling shows that the sensitivity of EG to DNA hybridization is controlled by changes in the resistance of the molecular layer as well as the space charge layer. The linear dynamic detection range of EG for DNA oligonucleotides is in the range of 5.0 × 10(-14) to 1 × 10(-6) M. In addition, with the use of differential pulse voltammetry, single stranded DNA, fully complimentary DNA, as well as single nucleotide polymorphisms can be differentiated on anodized EG by monitoring the oxidation signals of individual nucleotide bases.     

An addressable microelectrode array for electrochemical detection

Column and row electrodes on two different glass substrates were orthogonally arranged in order to assemble an addressable microelectrode device for the purpose of comprehensive electrochemical detection. Amperometric signal at the individual crossing point of the column and row electrodes was detected separately on the basis of redox cycling of localized electroactive species occurring between the electrodes. The addressable microelectrode device was simple and could be easily assembled; however, it comprised as many as 10 x 10 addressable detection points on a single chip. The basic electrochemical performance of the device was investigated by using the ferricyanide/ferrocyanide redox couple. Electrochemical responses at 100 individual points could be collected within 22 s. The present device was successfully used for imaging the spots of alkaline phosphatase on the array substrate. The results indicate that the device can be applied to comprehensive and high-throughput detection and imaging of biochemical species.