We describe a nanostructured immunosensor for the cardiovascular biomarker netrin 1. A glassy carbon electrode was consecutively modified with multi-walled carbon nanotubes (MWCNTs), nafion (to retain the MWCNTs), thionine-coated gold nanoparticles (Thi@AuNPs), and monoclonal antibodies against netrin 1. The modified electrode was characterized by transmission electron microscopy, cyclic voltammetry, differential pulse voltammetry, UV-visible spectrophotometry and X-ray diffraction. The presence of Thi@AuNPs warrants direct and convenient immobilization of the antibody. This immunoelectrode enables netrin 1 to be determined, best at a voltage of −300 mV (vs. SCE), with a limit of detection of 30 fg mL−1 (at an S/N ratio of 3) after a 50 min incubation time. The detection range extends from 0.09 to 1800 pg∙mL−1. The method is simple, sensitive, specific and reproducible. We presume this stable and reproducible biosensor to be useful for the early detection of cardiovascular diseases.
A high sensitivity immunoassay was developed for the detection of netrin 1 based on multi-walled carbon nanotubes, thionine and gold nanoparticles. Its excellent performance is ascribed to the good conductivity of MWCNTs and the combination of materials.
We describe an electrochemical bioassay for the detection of the activity of methyltransferase (MTase), and for screening this enzyme’s inhibitors. The assay is based on the conjugation of a hemin to a G-quadruplex that enables enzymatic signal amplification with the aid of exonuclease III (ExoIII). In the first step, double-stranded DNA containing the quadruplex-forming oligomer is assembled on the surface of a gold electrode and then methylated by DNA adenine methyltransferase (DAM). After cleaved by endonuclease DpnI, the methylated DNA is digested by ExoIII and the quadruplex-forming oligomers are liberated. This leads to the formation of a hemin/G-quadruplex (in presence of hemin and of potassium ions). The hemin/G-quadruplex catalyzes the oxidization of hydroquinone by H2O2 and the benzoquinone was formed to generate electrochemical signal. Finally, the gold electrode modified with reduced graphene oxide was used as working electrode for performing differential pulse voltammetry. The method has a detection limit of 0.31 unit · mL−1. A study on the inhibition of MTase showed it was inhibited by epicatechin with an IC50 value of 157 μM.
We describe an electrochemical bioassay for the detection of the activity of methyltransferase and for screening for its inhibitors. Due to the conjugation of a hemin to a G-quadruplex, strong enzymatic signal amplification is enabled with the aid of exonuclease III.
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.
Immunochromatography with secondary labeled antibodies caused 10-fold decrease of detection limit
We describe a turn-off fluorescence-based strategy for the detection of ATP by making use of aptamer-triggered dsDNA concatamers. This sensitive and easily controlled method is based on consecutive hybridization induced by ATP aptamers and their sectional complementary DNAs to form dsDNA concatamers. The intercalator SYBR Green I (SGI) was employed as a fluorescent probe. In the absence of ATP, the probe produces a strong signal. However, on addition of ATP, the binding of aptamer and ATP cause the concatamers to collapse and to release SGI whose fluorescence then is quenched. The effect was exploited to design a selective ATP assay by relating the decrease in fluorescence to the ATP concentration. A lower detection limit of 6.1 μM and a linear response in the 0 to 5000 μM concentration range was accomplished. The strategy was applied to cellular ATP assays, and the results obtained by this strategy and by the gold standard method are in good agreement. The method is sensitive, simple and cost efficient, and hence is promising in terms of future applications to determine ATP in cellular and other systems.
A turn-off fluorescence-based strategy for the selective detection of ATP by using aptamer-triggered dsDNA concatamers.
We report on the capillary electrophoretic behavior of citrate-capped gold and silver nanoparticles in aqueous medium when applying a ligand-exchange surface reaction with thiols. Gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) of similar size (39 ± 6 and 41 ± 7 nm, respectively) and shape were synthesized, covered with a citrate shell, and characterized by microscopic and spectroscopic techniques. The analysis of these NPs by CE was accomplished by using a buffer solution (pH 9.7; 40 mM SDS, 10 mM CAPS; 0.1 % methanol) containing the anions of thioctic acid or thiomalic acid. These are capable of differently interacting with the surface of the AuNPs and AgNPs and thus introducing additional negative charges. This results in different migration times due to the formation of differently charged nanoparticles.
Capillary electrophoretic behavior of citrate-capped gold and silver nanoparticles (NPs) in aqueous medium when applying a ligand-exchange surface reaction with thiols (thioctic and thiomalic acids), which introduces additional negative charges, has been studied
This article describes a fluorescent molecularly imprinted polymer (MIP) capable of selective fluorescent turn-on recognition of the tumor biomarker α-fetoprotein. The technique is making use of amino-modified Mn-doped ZnS quantum dots (QDs) as solid supports, 4-vinylphenylboronic acid and methyl methacrylate as the functional monomers, γ-methacryloxypropyl trimethoxysilane as the grafting agent, and α-fetoprotein as a template. A graft imprint is created on the surface of the QDs. The functional monomers are shown to play an important role in the formation of the binding sites and in preventing nonspecific protein binding. The resulting MIP-QDs display a good linear response to α-fetoprotein in the 50 ng · L−1 to 10 μg · L−1 concentration range, and the limit of detection is 48 ng · L−1. In our perception, the method has a wide scope in that it may be adapted to various other glycoproteins.
Schematic illustration of the synthesis of the MIP-QDs composites
This article describes an electrochemical immunosensor for rapid determination of Salmonella pullorum and Salmonella gallinarum. The first step in the preparation of the immunosensor involves the electrodeposition of gold nanoparticles used for capturing antibody and enhancing signals. In order to generate a benign microenvironment for the antibody, the ionic liquid (IL) 1-butyl-3-methylimidazolium hexafluorophosphate was used to modify the surface of a screen-printed carbon electrode (SPCE). The single steps of modification were monitored via cyclic voltammetry and electrochemical impedance spectroscopy. Based on these findings, a sandwich immunoassay was worked out for the two Salmonella species by immobilizing the respective unlabeled antibodies on the SPCE. Following exposure to the analytes, secondary antibody (labeled with HRP) is added to form the sandwich. After adding hydrogen peroxide and thionine, the latter is oxidized and its signal measured via CV. A linear response to the Salmonella species is obtained in the 104 to 109 cfu · mL−1 concentration range, and the detection limits are 3.0 × 103 cfu · mL−1 for both species (at an SNR of 3). This assay is sensitive, highly specific, acceptably accurate and reproducible. Given its low detection limit, it represents a promising tool for the detection of S. pullorum, S. gallinarum, and - conceivably - of other food-borne pathogens by exchanging the antibody.
We describe an electrochemical sandwich assay based on a screen-printed carbon electrode, gold nanoparticles and ILs and capable of detecting Salmonella pullorum and Salmonella gallinarum. The preparation is outlined in the Schematic.
We report on a facile immunoassay for porcine circovirus type 2 (PCV2) based on surface enhanced Raman scattering (SERS) using multi-branched gold nanoparticles (mb-AuNPs) as substrates. The mb-AuNPs in the immunosensor act as Raman reporters and were prepared via Tris base-induced reduction and subsequent reaction with p-mercaptobenzoic acid (pMBA). They possess good stability and high SERS activity. Subsequently, the modified mb-AuNPs were covalently conjugated to the monoclonal antibody (McAb) against the PCV2 cap protein to form SERS immuno nanoprobes. These were captured in a microtiterplate via a immunoreaction in the presence of target antigens. The effects of antibody concentration, reaction time and temperature on the sensitivity of the immunoassay were investigated. Under optimized assay conditions, the Raman signal intensity at 1,076 cm−1 increases logarithmically with the concentrations of PCV2 in the concentration ranging from 8 × 102 to 8 × 106 copies per mL. The limit of detection is 8 × 102 copies per mL. Compared to conventional detecting methods such as those based on PCR, the method presented here is rapid, facile and very sensitive.
We report on an electrochemical method for the determination of the activity of trypsin. A multi-functional substrate peptide (HHHAKSSATGGC-HS) is designed and immobilized on a gold electrode. The three His residues in the N-terminal are able to recruit thionine-loaded graphene oxide (GO/thionine), a nanocover adopted for signal amplification. Once the peptide is cleaved under enzymatic catalysis by trypsin (cleavage site: Lys residue), the His residues leave the electrode, and the GO/thionine cannot cover the peptide-modified electrode anymore. Thus, the changes of the electrochemical signal of thionine, typically acquired at a voltage of -0.35 V, can be used to determine the activity of trypsin. A detection range of 1 × 10−4 to 1 U, with a detection limit of 3.3 × 10−5 U, can be achieved, which is better than some currently available methods. In addition, the method is highly specific, facile, and has the potential for the detection of trypsin-like proteases.
Graphene oxide was adopted as a nanocover for the development of a sensitive electrochemical method to detect the activity of trypsin.
We describe the electrochemical preparation of bismuth nanoribbons (Bi-NRs) with an average length of 100 ± 50 nm and a width of 10 ± 5 μm by a potentiostatic method. The process occurs on the surface of a glassy carbon electrode (GCE) in the presence of disodium ethylene diamine tetraacetate that acts as a scaffold for the growth of the Bi-NRs and also renders them more stable. The method was applied to the preparation of Bi-NRs incorporated into reduced graphene oxide. This nanocomposite was loaded with the enzyme glucose oxidase onto a glassy carbon electrode. The resulting biosensor displays an enhanced redox peak for the enzyme with a peak-to-peak separation of about 28 mV, revealing a fast electron transfer at the modified electrode. The loading of the GCE with electroactive GOx was calculated to be 8.54 × 10−10 mol∙cm−2, and the electron transfer rate constant is 4.40 s−1. Glucose can be determined (in the presence of oxygen) at a relatively working potential of −0.46 V (vs. Ag|AgCl) in the 0.5 to 6 mM concentration range, with a 104 μM lower detection limit. The sensor also displays appreciable repeatability, reproducibility and remarkable stability. It was successfully applied to the determination of glucose in human serum samples.
A potentiostatic method was used to prepare reduced graphene oxide and bismuth nanoribbons nanocomposite on a glassy carbon electrode. This nanocomposite was loaded with enzyme glucose oxidase to fabricate a glucose biosensor.
High molecular-weight silk peptide (SP) was used to functionalize the surface of nanosheets of reduced graphene oxide (rGO). The SP-rGO nanocomposite was then mixed with mouse anti-human prostate specific antigen monoclonal antibody (anti-PSA) and coated onto a glassy carbon electrode to fabricate an immunosensor. By using the hexacyanoferrate redox system as electroactive probe, the immunosensor was characterized by voltammetry and electrochemical impedance spectroscopy. The peak current, measured at the potential of 0.24 V (vs. SCE), is distinctly reduced after binding prostate specific antigen (PSA). Response (measured by differential pulse voltammetry) is linearly related to PSA concentration in the range from 0.1 to 5.0 ng · mL−1 and from 5.0 to 80.0 ng∙mL−1, and the detection limit is 53 pg∙mL−1 (at an SNR of 3). The immunosensor was successfully applied to the determination of PSA in clinical serum samples, and the results were found to agree well with those obtained with an enzyme-linked immunosorbent assay.
Nanosheets of reduced graphene oxide were functionalized with silk peptide and used to immobilize anti-PSA to fabricate an immunosensor for PSA.
A simple, sensitive and accurate method was developed for solid-phase extraction and preconcentration of trace levels of gold in various samples. It is based on the adsorption of gold on modified oxidized multi-walled carbon nanotubes prior to its determination by graphite furnace atomic absorption spectrometry. The type and volume of eluent solution, sample pH value, flow rates of sample and eluent, sorption capacity and breakthrough volume were optimized. Under these conditions, the method showed linearity in the range of 0.2–6.0 ng L−1 with coefficients of determination of >0.99 in the sample. The relative standard deviation for seven replicate determinations of gold (at a level of 0.6 ng L−1) is ±3.8 %, the detection limit is 31 pg L−1 (in the initial solution and at an S/N ratio of 3; for n = 8), and the enrichment factor is 200. The sorption capacity of the modified MWCNTs for gold(III) is 4.15 mg g−1. The procedure was successfully applied to the determination of gold in (spiked) water samples, human hair, human urine and standard reference material with recoveries ranging from 97.0 to 104.2 %.
A sorbent based on modified carbon nanotubes was prepared and used to extract gold ion from various samples prior to its determination by graphite furnace atomic absorption spectrometry
A molecularly imprinted polymer (MIP) was prepared by self-polymerization of dopamine in the presence of bovine hemoglobin (BHb) and then deposited on the surface of an electrode modified with gold nanoparticles (AuNPs). Scanning electron microscopy, cyclic voltammetry, and differential pulse voltammetry were employed to characterize the modified electrode using the hexacyanoferrate redox system as an electroactive probe. The effects of BHb concentration, dopamine concentration, and polymerization time were optimized. Under optimized conditions, the modified electrode selectively recognizes BHb even in the presence of other proteins. The peak current for hexacyanoferrate, typically measured at + 0.17 V (vs. SCE), depends on the concentration of BHb in the 1.0 × 10−11 to 1.0 × 10−2 mg mL−1 range. Due to the ease of preparation and tight adherence of polydopamine to various support materials, the present strategy conceivably also provides a platform for the recognition and detection of other proteins.
Gold nanoparticles and molecularly imprinted self-polymerization dopamine were modified on gold electrode surface to recognize and determine bovine hemoglobin. Under the optimized conditions, the modified electrode showed specific adsorption, selective recognition, and sensitive detection of bovine hemoglobin.
We describe a simple and homogenous fluorimetric method for sensitive determination of DNA. It is based on target-triggered isothermal cycling and a cascade exponential amplification reaction that generates a large amount of a G-quadruplex. This results in strong fluorescence signal when using thioflavin T as a G-quadruplex-specific light-up fluorescent probe. Tedious handling after amplification is widely eliminated by the addition of thioflavin T. No other exogenous reagent is required. This detection platform is inexpensive and rapid, and displays high sensitivity for target DNA, with a detection limit as low as 91 pM.
The addition of target DNA can trigger the isothermal exponential amplification reaction to generate a large amount of G-quadruplex sequence oligonucleotides and then employ thioflavin T (Th T) (a G-quadruplex-specific light-up dye) as signal output for sensitive DNA detection.
Microparticles were synthesized by suspension copolymerization of the synthetic ionic liquid (IL) 1-allyl-3-methyl-imidazolium bromide with ethylene glycol dimethacrylate. The particles have a regular spherical shape and an average diameter of 65 ± 24 μm. Their affinity for the fluoroquinolone antibiotics ofloxacin (OFL), lomefloxacin (LOM) and ciprofloxacin (CIP) is much higher than that of the blank polymer (not containing an IL), of polymers using methacrylic acid as functional monomer, of hydrophilic-lipophilic balanced sorbents, and of C18 sorbents. The microparticles were applied to the solid-phase extraction and rapid preconcentration of the fluoroquinolones from urine which then were quantified by HPLC. The calibration plot covers the 0.05 to 20 μg mL−1 concentration range, and the average recoveries at three spiking levels range from 93.6 to 103.7 %, with RSD of ≤5.7 %. The method was successfully applied to the determination of fluoroquinolones in spiked urine.
Microparticles covalently functionalized with an ionic liquid ([Amim][Br]) were synthesized by suspension copolymerization and show higher affinity for fluoroquinolones than other sorbents. The microparticles were used as a sorbent for solid-phase extraction and preconcentration of three fluoroquinolones from urine.
We describe a method for the preparation of water-soluble gold nanoclusters (Au-NCs) from chloroauric acid using denatured-casein as both a reducing and stabilizing agent. The resulting Au-NCs were characterized by photoluminescence, UV–vis absorption, and X-ray photoelectron spectroscopies, and by transmission electron microscopy. The Au-NCs have an average diameter of 1.7 ± 0.2 nm and exhibit orange-red fluorescence emission peaking at 600 nm (with a Stokes’ shift as large as 237 nm), a quantum yield of 4.3 %, and good stability over the physiologically relevant range of pH values and ionic strength. Cytotoxicity studies showed the Au-NCs to display negligible effects in terms of altering cell proliferation or triggering apoptosis. Fluorescence imaging of HeLa cancer cells was accomplished by loading such cells with the Au-NCs. The fluorescence of the Au-NCs is found to be strongly quenched by Hg(II) ions, and thus the Au-NCs can be used for detecting and, possibly, imaging of Hg(II). An assay was worked out for the determination of Hg(II), and its limit of detection is 1.83 nM, which is 5.5 times lower than the maximum allowed concentration of Hg(II) in drinking water as defined by the US EPA.
Denatured-casein was firstly used as a reductant and stabilizing agent in facile preparation of orange-red fluorescent AuNCs for imaging of HeLa cells and for the quantitation of mercury(II)
We describe a sensitive and selective biosensor for the environmental metabolite 2-hydroxyfluorene (2-HOFlu). It is based on electrochemical impedance spectroscopy and was obtained by assembling a thiolated single-stranded DNA on a gold electrode via S-Au covalent bonding. It is then transformed to a K+-stabilized G-quadruplex-hemin complex which exhibits peroxidase-like activity to catalyze the oxidation of 2-HOFlu by H2O2. This results in the formation of insoluble products that are precipitated on the gold electrode. As a result, the charge transfer resistance (R CT) between the solution and the electrode surface is strongly increased within 10 min as demonstrated by using the ferro/ferricyanide system as a redox probe. The difference in the charge transfer resistances (ΔR CT) before and after incubation of the DNA film with 2-HOFlu and H2O2 serves as the signal for the quantitation of 2-HOFlu with a 1.2. nM detection limit in water of pH 7.4. The assay is highly selective over other selected fluorene derivatives. It was exploited to determine 2-HOFlu in spiked lake water samples where it displayed a detection limit of 3.6 nM. Conceivably, this method has a wide scope in that it may be applied to other analytes for which respective G-quadruplexes are available.
A G-quadruplex DNAzyme based impedimetric biosensor for sensitive detection of 2-hydroxyfluorene using hemin as a peroxidase enzyme mimic was constructed with a detection limit of 1.2 nM in water and 3.6 nM in spiked lake water samples.
Titanium dioxide nanorods (TNR) were grown on a titanium electrode by a hydrothermal route and further employed as a supporting matrix for the immobilization of nafion-coated horseradish peroxidase (HRP). The strong electrostatic interaction between HRP and TNR favors the adsorption of HRP and facilitates direct electron transfer on the electrode. The electrocatalytic activity towards hydrogen peroxide (H2O2) was investigated via cyclic voltammetry and amperometry. The biosensor exhibits fast response, a high sensitivity (416.9 μA·mM−1), a wide linear response range (2.5 nM to 0.46 mM), a detection limit as low as 12 nM, and a small apparent Michaelis-Menten constant (33.6 μM). The results indicate that this method is a promising technique for enzyme immobilization and for the fabrication of electrochemical biosensors.
A TiO2 nanorod film was directly grown on Ti substrate by a hydrothermal route, and was further employed for a supporting matrix to immobilize horseradish peroxidase as a biosensor electrode. The as-prepared hydrogen peroxide biosensor based on Nafion/HRP/TNR/Ti electrode exhibited fast response and excellent electrocatalytic activity toward H2O2, i.e., a high sensitivity (416.9 μA mM−1), a wide linear range (2.5 × 10−8 to 4.6 × 10−4 M) with a low detection limit (0.012 μM) and a small apparent Michaelis-Menten constant (33.6 μM).
Multilayered reduced graphene oxide (rGO) was functionalized with amino groups by treatment with nitrogen plasma. Raman spectroscopy showed plasma treatment not to substantially alter the chemical structure of rGO and that a wide range of functional nitrogen groups is evenly incorporated into the carbon lattice. The amino-modified rGO was used to design an electrochemical biosensor in which a DNAzyme, substrate DNA and Pb(II) and Hg(II) binding DNA were immobilized on the amino-rGO placed on a gold electrode. The high concentration of amino groups and the rough surface of the rGO favor DNA immobilization. Heavy metal ions are bound to the surface via specific interaction between DNA and the two ions which are detected by electrochemical impedance spectroscopy at a potential of 0.2 V (vs. Ag/AgCl). The detection limits for Pb(II) and Hg(II) are as low as 7.8 and 5.4 pM, respectively, and the analytical ranges extend from 0.01 to 100 nM. The sensor is highly specific and stable and therefore represents a highly promising tool for use in environmental monitoring.
A nanofilm of reduced graphene oxide was first modified with amino groups by treatment with nitrogen plasma. A special DNA was then anchored to the surface to obtain a biosensor for simultaneous detection of Pb(II) and Hg(II). The sensor has detection limits as low as 7.8 and 5.4 pM and is highly selective.
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.
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