Electrochemical immunoassay for the prostate specific antigen using ceria mesoporous nanospheres

We report on a sensitive electrochemical immunoassay for the prostate specific antigen (PSA). An immunoelectrode was fabricated by coating a glassy carbon electrode with multiwalled carbon nanotubes, poly(dimethyldiallylammonium chloride), CeO₂ and PSA antibody (in this order) using the layer-by-layer method. The immunosensor is then placed in a sample solution containing PSA and o-phenylenediamine (OPD). It is found that the CeO₂ nanoparticles facilitate the electrochemical oxidation of OPD, and this produces a signal for electrochemical detection of PSA that depends on the concentration of PSA. There is a linear relationship between the decrease in current and the concentration of PSA in the 0.01 to 1,000 pg mL^?1 concentration range, and the detection limit is 4 fg mL^?1. The assay was successfully applied to the detection of PSA in serum samples. This new differential pulse voltammetric immunoassay is sensitive and acceptably precise, and the fabrication of the electrode is well reproducible. Figure

A novel electrochemical immunoassay for prostate specific antigen (PSA) was developed. Ceria (CeO₂) mesoporous nanospheres facilitated the electrochemical oxidation of o-phenylenediamine (OPD). The developed immunoassay has high sensitivity and can be successfully applied for the detection of PSA in serum samples     

Mesoporous ZnO-NiO architectures for use in a high-performance nonenzymatic glucose sensor

Mesoporous ZnO-NiO architectures were prepared by thermal annealing of zinc-nickel hydroxycarbonate composites. The resulting architectures are shown to be assembled by many mesoporous nanosheets, and this results in a large surface area and a strong synergy between the ZnO and NiO nanoparticles. The material obtained by annealing at 400 °C was used as an electrode that responds to glucose over a wide concentration range (from 0.5 μM to 6.4 mM), with a detection limit as low as 0.5 μM, fast response time (<3 s), and good sensitivity (120.5 μA?·?mM^?1?·?cm^?2). Figure

The mesoporous ZnO-NiO architecture by annealing at 400 °C was used as an electrode that responds to glucose over a wide concentration range (from 0.5 μM to 6.4 mM), with a detection limit as low as 0.5 μM, fast response time (<3 s), and good sensitivity (120.5 μA?·?mM^?1?·?cm^?2      

Sensitive detection of enteropathogenic E. coli using a bfpA gene-based electrochemical sensor

We have developed a sensitive assay for enteropathogenic E. coli (EPEC) by integrating DNA extraction, specific polymerase chain reaction (PCR) and DNA detection using an electrode modified with the bundle-forming pilus (bfpA) structural gene. The PCR amplified products are captured on the electrode and hybridized with biotinylated detection probes to form a sandwich hybrid containing two biotinylated detection probes. The sandwich hybridization structure significantly combined the numerous streptavidin alkaline phosphatase on the electrode by biotin-streptavidin connectors. Electrochemical readout is based on dual signal amplification by both the sandwich hybridization structure and the enzyme. The electrode can satisfactorily discriminate complementary and mismatched oligonucleotides. Under optimal conditions, synthetic target DNA can be detected in the 1 pM to 10 nM concentration range, with a detection limit of 0.3 pM. EPEC can be quantified in the 10 to 10⁷ CFU mL^?1 levels within 3.5 h. The method also is believed to present a powerful platform for the screening of pathogenic microorganisms in clinical diagnostics, food safety and environmental monitoring.

Use of gold nanoparticle-labeled secondary antibodies to improve the sensitivity of an immunochromatographic assay for aflatoxin B1

We describe a sensitive method for the immunochromatographic determination of aflatoxin B1. It is based on the following steps: 1) Competitive interaction between non-labeled specific primary antibodies and target antigens in a sample and in the test zone of a membrane; 2) detection of the immune complexes on the membrane by using a secondary antibodies labeled with gold nanoparticles. The method enables precise adjustment of the required quantities of specific antibodies and the colloidal (gold) marker. It was applied in a lateral flow format to the detection of aflatoxin B1 and exhibits a limit of detection (LOD) of 160 pg?·?mL^?1 if detected visually, and of 30 pg?·?mL^?1 via instrumental detection. This is significantly lower than the LOD of 2 ng?·?mL^?1 achieved by conventional lateral flow analysis using the same reagents. Figure

Immunochromatography with secondary labeled antibodies caused 10-fold decrease of detection limit     

Lipoprotein(a) determination in human serum using a nitrilotriacetic acid derivative immunosensing scaffold on disposable electrodes

A novel strategy for the construction of a disposable integrated amperometric immunosensor for the sensitive and rapid determination of lipoprotein(a) (Lp(a)), an important predictor of cardiovascular disease risk, in human serum is reported. The approach uses a sandwich format involving the covalent immobilization of selective capture antibodies (antiLp(a)) on the surface of N-[N _α,N _α-bis(carboxymethyl)-lysine]-12-mercaptododecanamide (HS-NTA)-modified screen-printed carbon electrodes (SPCEs). After a blocking step with skimmed milk, the modified antiLp(a)-SPCEs were incubated with a mixture solution containing the target analyte and a fixed concentration of a specific biotinylated antibody (biotin-antiLp(a)) and a streptavidin-horseradish peroxidase (HRP) (Strep-HRP) conjugate. The amperometric responses of the resulting immunosensor at ?0.10 V (vs an Ag pseudo-reference electrode), upon addition of 3,3′,5,5′-tetramethylbenzidine (TMB) as electron transfer mediator and H₂O₂ as the enzyme substrate, were used to monitor the extent of the immunoreactions. The developed methodology exhibited a wide range of linearity between 0.02 and 10 μg mL^?1, a low detection limit (LOD) of 8 ng mL^?1, and a great selectivity against other serum components. The usefulness of the Lp(a) immunosensor was demonstrated by analyzing spiked serum samples as well as a reference serum containing a certified Lp(a) content. Figure

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Nanostructured platform for the detection of Neisseria gonorrhoeae using electrochemical impedance spectroscopy and differential pulse voltammetry

We report on a nanocomposite based genosensor for the detection of Neisseria gonorrhoeae, a bacterium causing the sexually transmitted disease gonorrhoea. Amino-labeled probe DNA was covalently immobilized on electrochemically prepared polyaniline and iron oxide (PANI-Fe₃O₄) nanocomposite film on an indium tin oxide (ITO) electrode. Scanning electron microscopy, transmission electron microscopy, electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) techniques have been employed to characterize surface of the modified electrode. The genosensor has detection limits of 1?×?10^-15 M and 1?×?10^-17 M, respectively, using the EIS and DPV techniques. This biosensor can discriminate a complementary sequence from a single-base mismatch and from non-complementary DNA, and has been utilized for detection of DNA extracted from N. gonorrhoeae culture, and from patient samples with N. gonorrhoeae. It is found to exhibit good specificity for N. gonorrhoeae species and shows no response towards non-gonorrhoeae type of Neisseria species (NgNs) and other gram-negative bacterias (GNBs). The affinity constant for hybridization calculated using the Langmuir adsorption isotherm model is found to be 3.39?×?10⁸ M^-1.

Evaluation of ultrahigh-performance liquid chromatography columns for the analysis of unmodified and antisense oligonucleotides

Ultrahigh-performance liquid chromatography has been used for the separation and analysis of unmodified and modified antisense oligonucleotides. For this reason, we tested various columns of low particle sizes in our analysis of unmodified and phosphorothioate oligonucleotides. The influence of both the type and concentration of ion-pair reagent on the retention of the studied biomolecules was tested. The developed methods were used for separation of unmodified oligonucleotides and to determine antisense oligonucleotides in human serum samples. The results proved that octadecyl and phenyl columns are the most selective in the resolution of oligonucleotides which differ in the position of single nucleotides in the sequence. The phenyl column was selected and applied for the analysis of phosphorothioate oligonucleotides in serum samples. The calibration plots showed good linearity within the test concentration ranges. The intra-day CV of the calibration curve slopes was in the range of 1.6 to 4.2 %. The limits of detection (LODs) were in the range of 0.11–0.16 μg mL^?1, while the limit of quantification (LOQ) values were between 0.35 and 0.51 μg mL^?1. Figure

Determination of antisense phosphorothioate oligonucleotides in serum     

Fluorometric determination of paraoxon in human serum using a gold nanoparticle-immobilized organophosphorus hydrolase and coumarin 1 as a competitive inhibitor

A dimeric organophosphorus hydrolase (OPH; EC 3.1.8.1; 72 kDa) was isolated from wild-type bacteria, analyzed for its 16s rRNA sequence, purified, and immobilized on gold nanoparticles (AuNPs) to form the transducer part of a biosensor. The isolated strain was identified as Pseudomonas aeruginosa. The AuNPs were characterized by transmission electron microscopy and localized surface plasmon resonance. Covalent binding of OPH to the AuNPs was confirmed by spectrophotometry, enzymatic activity assays, and FTIR spectroscopy. Coumarin 1, a competitive inhibitor of OPH, was used as a fluorogenic probe. The bioconjugates quench the emission of coumarin 1 upon binding, but the addition of paraoxon results in an enhancement of fluorescence that is directly proportional to the concentration of paraoxon. The gold-OPH conjugates were then used to determine paraoxon in serum samples spiked with varying levels of paraoxon. The method works in the 50 to 1,050 nM concentration range, has a low standard deviation (with a CV of 5.7–11 %), and a detection limit as low as 5?×?10^?11 M.

A magnetic metal-organic framework as a new sorbent for solid-phase extraction of copper(II), and its determination by electrothermal AAS

We report on the synthesis of Fe₃O₄-functionalized metal-organic framework (m-MOF) composite from Zn(II) and 2-aminoterephthalic acid by a hydrothermal reaction. The magnetic composite is iso-reticular and was characterized by FTIR, X-ray diffraction, SEM, magnetization, and TGA. The m-MOF was then applied as a sorbent for the solid-phase extraction of trace levels of copper ions with subsequent quantification by electrothermal AAS. The amount of sorbent applied, the pH of the sample solution, extraction time, eluent concentration and volume, and desorption time were optimized. Under the optimum conditions, the enrichment factor is 50, and the sorption capacity of the material is 2.4 mg g^?1. The calibration plot is linear over the 0.1 to 10 μg L^?1 Cu(II) concentration range, the relative standard deviation is 0.4 % at a level of 0.1 μg L^?1 (for n?=?10), and the detection limit is as low as 73 ng L^?1. We consider this magnetic MOF composite to be a promising and highly efficient material for the preconcentration of metal ions.

A sensitive immunosorbent bio-barcode assay based on real-time immuno-PCR for detecting 3,4,3',4'-tetrachlorobiphenyl

A functionalized gold-nanoparticle bio-barcode assay, based on real-time immuno-PCR (IPCR), was designed for the determination of 3,4,3',4'-tetrachlorobiphenyl (PCB77). 15 nm gold nanoparticles were synthesized, and modified with thiol-capped DNA and goat anti-rabbit IgG. The nanoparticle probes were used to replace antibody–DNA conjugate in the IPCR, and were fixed on the PCR tube wall via the immune reaction. Real-time PCR was performed to quantify the DNA signal directly. Under optimized conditions, the new method was used to detect PCB77 with a linearity range from 5 pg L^?1 to 10 ng L^?1, and the limit of detection (LOD) was 1.72 pg L^?1. Real samples of Larimichthys polyactis, collected from the East China Sea, were analyzed. Recovery was from 82 % to 112 %, and the coefficient of variation (CV) was acceptable. The results were compared with GC–ECD, revealing that the method would be acceptable for providing rapid, semi-quantitative, and reliable test results for making environmental decisions.

Visual detection and microplate assay for Staphylococcus aureus based on aptamer recognition coupled to tyramine signal amplification

We have developed a specific method for the visual detection of Staphylococcus aureus based on aptamer recognition coupled to tyramine signal amplification technology. A biotinylated aptamer specific for S. aureus was immobilized on the surface of the wells of a microplate via biotin-avidin binding. Then, the target bacteria (S. aureus), the biotinylated-aptamer-streptavidin-HRP conjugates, biotinylated tyramine, hydrogen peroxide and streptavidin-HRP were successively placed in the wells of the microplate. After adding TMB reagent and stop solution, the intensity of the yellow reaction product can be visually inspected or measured with a plate reader. Under optimized conditions, there is a linear relationship between absorbance at 450 nm and the concentration of S. aureus in the 10 to 10⁷ cfu mL^?1 concentration range (with an R² of 0.9976). The limit of detection is 8 cfu mL^?1.

Fluorogenic assays for activated protein C using aptamer modified magnetic beads

We describe a fluorogenic assay for activated protein C (APC) by using magnetic beads modified with DNA aptamers, taking advantage of strong binding affinity of aptamer, facile magnetic separation, and signal amplification via an enzymatic reaction. APC is specifically captured from a sample by the DNA aptamers on magnetic beads, and the concentrated APC then catalyzes the conversion of a fluorogenic substrate of APC to a fluorescent product. Detection of APC is achieved by measuring the generated product. This method is simple, sensitive, and specific. APC can be detected at 0.4 pM concentration level in a sample volume of 250 μL, corresponding to 0.1 femtomole of APC, when 2-h enzymatic reaction is employed. The proteins thrombin, trypsin, proteinase K, chymotrypsin, and elastase do not interfere.

Selection and identification of ssDNA aptamers recognizing zearalenone

Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin produced by Fusarium graminearum on maize and barley. Because most current methods of ZEN detection rely on the use of low-stability antibodies or expensive equipment, we sought to develop a rapid, low-cost determination method using aptamers instead of antibodies as the specific recognition ligands. This work describes the isolation and identification of single-stranded DNA (ssDNA) aptamers recognizing ZEN using the modified systematic evolution of ligands by exponential enrichment methodology based on magnetic beads. After 14 rounds of repeated selection, a highly enriched ssDNA library was sequenced and 12 representative sequences were assayed for their affinity and specificity. The best aptamer, 8Z₃₁, with a dissociation constant (K _d) of 41?±?5 nM, was successfully applied in the specific detection of ZEN in binding buffer and in real samples based on a magnetic separation/preconcentration procedure. This analytical method provided a linear range from 3.14?×?10^?9 to 3.14?×?10^?5 M for ZEN, and the detection limit was 7.85?×?10^?10 M. The selected aptamers are expected to be used in the potential development of affinity columns, biosensors, or other analytical systems for the determination of ZEN in food and agricultural products.

A nanocomposite consisting of plasma-polymerized propargylamine and graphene for use in DNA sensing

We report on a novel graphene-based nanoarchitecture modified with plasma-polymerized propargylamine (G-PpPG) and its application in electrochemical sensors for DNA. Films of G-PpPG were characterized by X-ray photoelectron spectroscopy and electrochemical impedance spectroscopy. The presence of graphene enhances the electrochemical activity of the films, and the high density of amino groups (deposited at a low plasma input power) on their surface assists in the immobilization of probe DNA on the water-swollen polymeric network. By contrast, the degree of hybridization of the total complementary target DNA to the probe DNA remains unchanged when G-PpPG nanofilms prepared at higher input power. No substantial non-specific adsorption of totally mismatched target DNA on the polymer films is observed because of the complete coverage of the probe DNA. The detection limit for total complementary target DNA is approximately 1.84 nmol?·?L^?1. The dynamic range extends from 0.1 to 1,000 nmol?·?L^?1. The new nanocomposite may also be used to immobilize other probe DNA sequences, and this makes the approach potentially applicable to the detection of other oligomers. Figure

Preparing the DNA sensor made from the graphene-based nanoarchitecture modified by using PpPG (G-PpPG) includes the following processes: (a) Modifying the Au electrode with the graphene nanosheet, (b) depositing the PpPG film onto the Au electrode coated with graphene, (c) immobilizing the probe DNA onto the G-PpPG film, and (d) hybridizing the MM0 target with the G-PpPG film immobilized with P1     

Cetyltrimethylammonium-coated magnetic nanoparticles for the extraction of bromate,followed by its spectrophotometric determination

We report on a combination of magnetic solid-phase extraction and spectrophotometric determination of bromate. Cetyltrimethylammonium ion was adsorbed on the surface of phenyl-functionalized silica-coated Fe₃O₄ nanoparticles (Ph-SiO₂@Fe₃O₄), and these materials served as the sorbent. The effects of surfactant and amount of sorbent, the composition of the desorption solution, the extraction time and temperature were optimized. Under optimized conditions, an enrichment factor of 12 was achieved, and the relative standard deviation is 2.9 % (for n?=?5). The calibration plot covers the 1–50 ng mL^?1 range with reasonable linearity (r ²?>?0.998); and the limit of detection is 0.5 ng mL^?1. The method is not interfered by ionic compounds commonly found in environmental water samples. It was successfully applied to the determination of bromate in spiked water samples.

Determination of fluoroquinolone antibiotics by microchip capillary electrophoresis along with time-resolved sensitized luminescence of their terbium(III) complexes

We report on the time-resolved detection of the three fluoroquinolone (FQs) antibiotics ciprofloxacin (CIP), enrofloxacin (ENR) and flumequine (FLU). On addition of terbium(III) ions, the terbium(III)-FQs chelates are formed in-situ in an on-capillary derivatization reaction of a microfluidic system. The laser-induced terbium(III)-sensitized luminescence of the chelates is measured at excitation/emission wavelengths of 337/545 nm. The analytes can be separated and quantified within less than 4 min. A solid phase extraction step for analyte preconcentration can be included prior to chelation and microchip capillary electrophoresis. The analytical ranges of the calibration graphs for CIP, ENR and FLU are from 10.6 to 60.0, 10.3 to 51.0, and 11.5 to 58.8 ng mL^?1, respectively, and the detection limits are 3.2, 3.1 and 3.6 ng mL^?1, respectively. The precision was established at two concentration levels of each analyte and revealed relative standard deviations in the range from 3.0 to 10.2 %. The method was applied to the analysis of FQ-spiked water samples. Figure

We report on the time-resolved detection of the three fluoroquinolone antibiotics. The analytes can be separated and quantified within less than 4 min. A solid phase extraction step for analyte preconcentration can be included prior to chelation and microchip capillary electrophoresis.     

Hybrid nanonet/nanoflake NiCo2O4 electrodes with an ultrahigh surface area for supercapacitors

An integrated electrode consisting of hybrid nanonet/nanoflake NiCo₂O₄ grown on stainless steel mesh substrates exhibits a high specific capacitance while maintaining high-rate capability and good cycling stability. The specific capacitance reaches a maximum of 911 F g^?1 at a current density of 10 A g^?1, which can still retain 864 F g^?1 (94.8 % retention) after 10,000 cycles. These much-improved electrochemical performances are attributed to the unique architecture of NiCo₂O₄ electrode. The interconnected nanonet NiCo₂O₄ with an ultrahigh surface area significantly facilitates the rapid ion/electron transport and guarantees good mechanical adhesion, while the ultrathin nanoflakes further extend the active sites for fast redox reactions for efficient energy storage. Figure

Hybrid nanonet/nanoflake NiCo₂O₄ grown on stainless steel mesh exhibits superior capacitive performance and long-life stability as an integrated electrode for high-performance supercapacitors.     

Sensitive immunoassay for the β-agonist ractopamine based on glassy carbon electrode modified with gold nanoparticles and multi-walled carbon nanotubes in a film of poly-arginine

We are presenting an electrochemical immunosensor for the determination of the β-agonist and food additive ractopamine. A glassy carbon electrode (GCE) was modified with gold nanoparticles and a film of a composite made from poly(arginine) and multi-walled carbon nanotubes. Antibody against ractopamine was immobilized on the surface of the modified GCE which then was blocked with bovine serum albumin. The assembly of the immunosensor was followed by electrochemical impedance spectroscopy. Results demonstrated that the semicircle diameter increases, indicating that the film formed on the surface hinders electron transfer due to formation of the antibody-antigen complex on the modified electrode. Under optimal conditions, the peak current obtained by differential pulse voltammetry decreases linearly with increasing ractopamine concentrations in the 0.1 nmol?L^?1 to 1 μmol?L^?1 concentration range. The lower detection limit is 0.1 nmol?L^?1. The sensor displays good stability and reproducibility. The method was applied to the analysis of spiked swine feed samples and gave satisfactory results. Figure

Immunoassay for ractopamine based on glassy carbon electrode modified with gold nanoparticles and a film of a composite made from poly (arginine) and multi-walled carbon nanotubes was proposed. Under optimal conditions, the peak currents obtained by differential pulse voltammetry decreases linearly with increasing ractopamine concentrations in the 0.1 nmol?L^?1 to 1 μmol?L^?1 concentration range. The detection limit is 0.1 nmol?L^?1.     

Zundel-Type H-Bonding in Biomolecular Ions

Using quantum chemical calculations and infrared multiphoton dissociation (IRMPD) spectroscopy in the fingerprint and X-H stretching regions, we demonstrate here that the all-Ala b ₆ fragment ion features a macrocyclic structure with C₂ symmetry. For this structure, the ionizing proton is equally shared by the Ala(1) and Ala(4) amide oxygens in a Zundel-type symmetric (X…H⁺…X) H-bond. Figure

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Graphene ceramic composite as a new kind of surface-renewable electrode: application to the electroanalysis of ascorbic acid

This study introduces a new surface-renewable electrode based on a sol–gel derived graphene ceramic composite. The electrode was prepared by dispersing graphene nanosheets into a solution of the sol–gel precursors containing methyl triethoxysilane in methanol and hydrochloric acid. During hydrolysis of methyl triethoxysilane, the graphene nanosheets are trapped in the gel. After moulding and drying the composite, it can be used as a surface-renewable electrode to which we refer as a graphene ceramic composite electrode (GCCE). Cyclic voltammograms of the hexacyanoferrate(II/III) model redox system at the GCCE were compared to those obtained with a conventional carbon ceramic electrode and showed a highly improved electron transfer rate at the GCCE. The electrocatalytic oxidation of ascorbic acid as a model analyte was then studied at working potential of 50 mV and over the 3–84 μM concentration range. It revealed a sensitivity of 6.06 μA μM^?1 cm^?2 and a detection limit of 0.82 μM. The GCCE was successfully applied to the determination of ascorbic acid in orange juice and urine samples. Advantages such as good mechanical and chemical stability, ease of fabrication, and reproducible preparation make the GCCE a potentially useful and widely applicable renewable electrode for use in routine analysis. Fig. 1

(Left) FESEM image and photograph of the graphene ceramic composite electrode (GCCE); (right) the cyclic voltammogram of the renewable GCCE in 5 mM K₃[Fe(CN)₆] solution containing 0.1 M KNO₃ at scan rate of 100 mV s^?1