Ultrasensitive electrochemiluminescent immunoassay for morphine using a gold electrode modified with CdS quantum dots, polyamidoamine, and gold nanoparticles

We report on a novel electrochemiluminescent (ECL) immunoassay for the ultrasensitive determination of morphine by making use of a gold electrode which was modified with a nanocomposite film containing self-assembled polyamidoamine (PAMAM) CdS quantum dots and electrodeposited gold nanoparticles (Au-NPs). The highly uniform and well-dispersed quantum dots were capped with PAMAM dendrimers. Due to the synergistic effect of the modified quantum dots and the electrodeposited Au-NPs, the ECL response is dramatically enhanced. Under optimal experimental conditions, the immunoreaction between morphine and anti-morphine antibody resulted in a decrease of the ECL signal because of steric hindrance. The calibration plot is linear in the morphine concentration range from 0.2 to 180 ng?mL^?1, with a detection limit as low as 67 pg?mL^?1. The sensor was successfully applied to the determination of morphine in blood plasma. This kind of assay is expected to pave new avenues in label-free drug assays.

Electrodeposition of CdSe quantum dots and its application to an electrochemiluminescence immunoassay for α-fetoprotein

We report on the first label-free electrochemiluminescence (ECL) immunosensor for α-fetoprotein (AFP). It is based on the use of CdSe quantum dots that were electrodeposited directly on a gold electrode from an electrolyte (containing cadmium sulfate, EDTA and selenium dioxide) by cycling the potential between 0 and -1.2?V (vs. SCE) for 60?s. The electrodeposited dots were characterized by scanning electron microscopy and energy dispersive spectroscopy. Under optimal conditions, the specific immunoreaction between AFP and anti-AFP resulted in a decrease of the ECL signal because of the steric hindrance and the transfer inhibition by peroxodisulfate. The quenching effect of the immunoreaction on the intensity of the ECL was used to establish a calibration plot which is linear in the range from 0.05 to 200?ng?mL^?1. The detection limit is 2?pg?mL^?1. The assay is highly sensitive and satisfactorily reproducible. In our opinion it opens new avenues to apply ECL in label-free biological assays.

Biotinylation of quantum dots for application in fluoroimmunoassays with biotin-avidin amplification

A competitive microplate fluoroimmunoassay was developed for the determination of human serum albumin in urine. It is based on the use of biotinylated CdTe quantum dots (QDs) whose synthesis is optimised in terms of storage stability, purification, and signal-to-noise ratio. The bioconjugated QDs were characterised by gel chromatography and gel electrophoresis. Storage stability and quantum yield were investigated. The excitation/emission wavelengths are 360/620?nm. The immunoassay of human serum albumin in urine has a working range from 1.7 to 10?μg.mL^?1, and the limit of detection is 1.0?μg.mL^?1.

Electrochemiluminescent sensing of dopamine using CdTe quantum dots capped with thioglycolic acid and supported with carbon nanotubes

We have synthesized water-dispersible CdTe quantum dots (QDs) capped with thioglycolic acid. Their quantum yield is higher than 54%. A sensitive electrochemiluminescence (ECL) method was established based on the modification of the composite of the QDs, carbon nanotubes and chitosan on indium tin oxide glass. The sensor displays efficient and stable anodic ECL which is quenched by dopamine. A respective sensor was designed that responds to dopamine linearly in the range of 50?pM to 10?nM, and the detection limit is 24?pM. Dopamine was determined with this sensor in spiked cerebro-spinal fluid with average recoveries of 95.7%.

Highly sensitive electrochemiluminescence “turn-on” aptamer sensor for lead(II) ion based on the formation of a G-quadruplex on a graphene and gold nanoparticles modified electrode

We have developed a “turn on” model of an electrochemiluminescence (ECL) based assay for lead ions. It is based on the formation of a G-quadruplex from an aptamer labeled with quantum dots (QDs) and placed on an electrode modified with of graphene and gold nanoparticles (AuNPs). A hairpin capture probe was labeled with a thiol group at the 5′-end and with an amino group at the 3′-end. It was then self-assembled on the electrode modified with graphene and AuNPs. In the absence of Pb(II), the amino tag on one end of the hairpin probe is close to the surface of the electrode and therefore unable to interact with the QDs because of steric hindrance. The ECL signal is quite weak in this case. If, however, Pb(II) is added, the stem-loop of the aptamer unfolds to form a G-quadruplex. The amino group at the 3′-end will become exposed and can covalently link to a carboxy group on the surface of the CdTe QDs. This leads to strong ECL. Its intensity increases (“turns on”) with the concentration of Pb(II). Such a “turn-on” method does not suffer from the drawbacks of “turn-off” methods. ECL intensity is linearly related to the concentration of Pb(II) in the 10 p mol·L^?1 to 1 n mol·L^?1 range, with a 3.8 p mol·L^?1 detection limit. The sensor exhibits very low detection limits, good selectivity, satisfying stability, and good repeatability.

Ultrasensitive electrochemiluminescent detection of pentachlorophenol using a multiple amplification strategy based on a hybrid material made from quantum dots,graphene, and carbon nanotubes

We report on a highly sensitive and selective electrochemiluminescence (ECL) based method for the determination of pentachlorophenol (PCP). It is based on a new hybrid material composed of CdS quantum dots (QDs), graphene, and carbon nanotubes (CNTs), and uses peroxodisulfate as the coreactant. The use of this system results in a nearly 18-fold increase in ECL intensity. On interaction between PCP and the QDs, a decrease in ECL intensity is observed at PCP in a concentration as low as 1.0 pM and over a wide linear range (from 1.0 pM to 1.0 nM). The method is hardly affected by other chlorophenols and nitrophenols, and the electrode can be recycled.

Quantum dots on electrodes—new tools for bioelectroanalysis

The review covers recent developments in which quantum dots (QDs) are combined with electrodes for detection of analytes. Special focus will be on the generation of photocurrents and the possibility of spatially resolved, light-directed analysis. Different modes for combining biochemical reactions with QDs will be discussed. Other applications involve the use of QDs as labels in binding analysis. Different methods have been developed for read-out. In addition to photocurrent analysis, voltammetric detection of metals and electrochemiluminescence (ECL) can be used. In the latter, light is the sensor signal. ECL-based systems combine the advantage of very sensitive analytical detection with rather simple instrumentation.

Highly sensitive hybridization assay using the electrochemiluminescence of an ITO electrode, CdTe quantum dots functionalized with hierarchical nanoporous PtFe nanoparticles, and magnetic graphene nanosheets

We report on a disposable microdevice suitable for sandwich-type electrochemiluminescence (ECL) detection of DNA. The method is making use of CdTe quantum dots functionalized with hierarchical nanoporous PtFe (CdTe@PtFe) nanoparticles and with magnetic graphene nanosheets. The latter were selected as carriers for the capture DNA due to their excellent biomagnetic separation capability and electrical properties. The CdTe@PtFe nanoparticles were used to label the signal DNA which resulted in distinctly enhanced ECL owing to the large specific surface area and good electrical conductivity of the PtFe alloy. A DNA sensor was constructed on a disk-shaped indium tin oxide electrode that was fabricated via etching. Under optimal conditions, the biosensor responds linearly to DNA in the 0.02 fM to 5000 fM concentration range, with a detection limit as low as 15 aM. The electrode is regenerable. The method displays excellent specificity, extremely good sensitivity, and is highly reproducible.

One-step electrochemical immunosensing for simultaneous detection of two biomarkers using thionine and ferrocene as distinguishable signal tags

We report on a new kind of electrochemical immunosensors for simultaneous determination of the biomarkers carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). Thionine and ferrocene were applied as distinguishable electrochemical tags (and mediators) which were covalently conjugated on anti-AFP and anti-CEA antibodies, respectively, via carboxy groups. The resulting conjugates were co-immobilized on a glassy carbon electrode functionalized with gold nanoparticles. Finally, horseradish peroxidase (HRP) was immobilized onto the modified electrode. Labeled thionine and ferrocene, respectively, act as distinguishable tags for simultaneous determination of AFP and CEA due to the difference in the location of their voltammetric peaks. With a one-step immunoassay format, the analytes in the sample produced transparent immunoaffinity reaction with the corresponding antibodies on the electrode. Once the immunocomplex is formed, it partially inhibits the active center of the immobilized HRP, and this decreased the activity of HRP in terms of reduction of hydrogen peroxide. This immunosensor enables the simultaneous determination of AFP and CEA in a single run and within the same dynamic range (0.01–50?ng?mL^?1) and the same lower detection limit (0.01?ng?mL^?1). The reproducibility and stability of the immunosensors are acceptable. The dual immunosensor was applied to evaluate several specimens, and the assay results are in acceptable agreement with clinical data.

Quantum dot induced phototransformation of 2,4-dichlorophenol, and its subsequent chemiluminescence reaction

We have studied the CdTe quantum dot-induced phototransformation of 2,4-dichlorophenol (2,4-DCP) and its subsequent chemiluminescence (CL) reaction. Quantum dots (QDs) of different size and capped with thioglycolic acid were prepared and characterized by molecular spectroscopy, X-ray diffraction and transmission electron microscopy. In the presence of QDs, 2,4-DCP is photochemically transformed into a long-living light emitting precursor which can react with N-bromosuccinimide to produce CL with peak wavelengths at 475 and 550 nm. The formation of singlet oxygen during the phototransformation process was confirmed by the enhancement effect of deuterium oxide on the CL reaction and the change in the UV spectrum of a chemical trap. The CL intensity is linearly related to the concentration of 2,4-DCP in the range from 0.36 to 36 μmol L^?1, and the detection limit (at 3σ) is 0.13 μmol L^?1.

Determination of formaldehyde based on the enhancement of the chemiluminescence produced by CdTe quantum dots and hydrogen peroxide

Water-soluble cadmium telluride quantum dots (CdTe QDs) capped with glutathione (GSH) display chemiluminescence (CL) emission on reaction with hydrogen peroxide (H₂O₂) in strongly alkaline medium. It is found that the CL is strongly enhanced on addition of formaldehyde in aqueous solution. A flow injection system was developed, and it is shown that there is good linearity between CL intensity and the concentration of formaldehyde in the 0.06–3.0 μg L^?1 range. The limit of detection is as low as 10 ng L^?1. The method was successfully applied to the determination of formaldehyde in indoor air after adsorption into an aqueous phase. The recoveries for the real samples range from 97 % to 102.5 %, and the relative standard deviation is <3.8 % for intra- and inter-assay precision.

Highly sensitive detection of lead(II) ion using multicolor CdTe quantum dots

Multicolor and water-soluble CdTe quantum dots (QDs) were synthesized with thioglycolic acid (TGA) as stabilizer. These QDs have a good size distribution, display high fluorescence quantum yield, and can be applied to the ultrasensitive detection of Pb(II) ion by virtue of their quenching effect. The size of the QDs exerts a strong effect on sensitivity, and quenching of luminescence is most effective for the smallest particles. The quenching mechanism is discussed. Fairly selective detection was accomplished by utilizing QDs with a diameter of 1.6?nm which resulted in a detection limit of 4.7?nmol?L^?1 concentration of Pb(II). The method was successfully applied to the determination of Pb(II) in spinach and citrus leaves, and the results are in good agreement with those obtained with atomic absorption spectrometry.

Nanoporous gold as a solid support for protein immobilization and development of an electrochemical immunoassay for prostate specific antigen and carcinoembryonic antigen

Nanoporous gold (NPG) was utilized as a support for immobilizing alkaline phosphatase (ALP) conjugated to monoclonal antibodies against either prostate specific antigen (PSA) or carcinoembryonic antigen (CEA). The antibody-ALP conjugates were coupled to self-assembled monolayers of lipoic acid and used in direct kinetic assays. Using the enzyme substrate p-aminophenylphosphate, the product p-aminophenol was detected by its oxidation near 0.1?V (vs. Ag|AgCl) using square wave voltammetry. The difference in peak current arising from oxidation of p-aminophenol before and after incubation with biomarker increased with biomarker concentration. The response to these two biomarkers was linear up to 10?ng mL^?1 for CEA and up to 30?ng mL^?1 for PSA. The effect of interference on the PSA assay was studied using bovine serum albumin (BSA) as a model albumin protein. The effect of interference from a serum matrix was examined for the PSA assay using newborn calf serum. A competitive version of the immunoassay using antigen immobilized onto the NPG surface was highly sensitive at lower antigen concentration. Estimates of the surface coverage of the antibody-ALP conjugates on the NPG surface are presented.

Highly sensitive FRET-based fluorescence immunoassay for aflatoxin B1 using cadmium telluride quantum dots

We report on a competitive immunoassay for the determination of aflatoxin B1 using fluorescence resonance energy transfer (FRET) from anti-aflatoxin B1 antibody (immobilized on the shell of CdTe quantum dots) to Rhodamine 123 (Rho 123-labeled aflatoxin B1 bound to albumin). The highly specific immunoreaction between the antibody against aflatoxin B1 on the QDs and the labeled-aflatoxin B1 brings the Rho 123 fluorophore (acting as the acceptor) and the QDs (acting as the donor) in close spatial proximity and causes FRET to occur upon photoexcitation of the QDs. In the absence of unlabeled aflatoxin B1, the antigen-antibody complex is stable, and strong emission resulting from the FRET from QDs to labeled aflatoxin B1 is observed. In the presence of aflatoxin B1, it will compete with the labeled aflatoxin B1-albumin complex for binding to the antibody-QDs conjugate so that FRET will be increasingly suppressed. The reduction in the fluorescence intensity of the acceptor correlates well with the concentration of aflatoxin B1. The feasibility of the method was established by the detection of aflatoxin B1 in spiked human serum. There is a linear relationship between the increased fluorescence intensity of Rho 123 with increasing concentration of aflatoxin B1 in spike human serum, over the range of 0.1–0.6 μmol·mL^?1. The limit of detection is 2?×?10^?11 M. This homogeneous competitive detection scheme is simple, rapid and efficient, and does not require excessive washing and separation steps.

Detection of trace nitrite in waters using a QDs-based chemiluminescence analysis system

In the present work, a novel flow-injection chemiluminescence method based on CdTe quantum dots (QDs) was developed for the determination of nitrite. Weak chemiluminescence (CL) signals were observed from a CdTe QDs–H₂O₂ system under basic conditions. The addition of a trace amount of hemoglobin (Hb) caused the CL from the CdTe QDs–H₂O₂ system to increase substantially. In the presence of nitrite, the ferrous Hb reacted with the nitrate to form ferric Hb and NO. The NO then bound to ferrous Hb to generate iron nitrosyl Hb. As a result, the CL signal from the CdTe QDs–H₂O₂–Hb system was quenched. Thus, a flow-injection CL analytical system for the determination of trace nitrite was established. Under optimum conditions, there was a good linear relationship between CL intensity and the concentration of nitrite in the range 1.0?×?10^?9 to 8.0?×?10^?7 mol L^?1 (R ²?=?0.9957). The limit of detection for nitrite using this system was 3.0?×?10^?10 mol L^?1 (S/N?=?3). This method was successfully applied to detect nitrite in water samples.

Disposable immunoassay for hepatitis B surface antigen based on a graphene paste electrode functionalized with gold nanoparticles and a Nafion-cysteine conjugate

We report on the modification of a graphene paste electrode with gold nanoparticles (AuNPs) and a Nafion-L-cysteine composite film, and how this electrode can serve as a platform for the construction of a novel electrochemical immunosensor for the detection of hepatitis B surface antigen (HBsAg). To obtain the immunosensor, an antibody against HBsAg was immobilized on the surface of the electrode, and this process was followed by cyclic voltammetry and electrochemical impedance spectroscopy. The peak currents of a hexacyanoferrate redox system decreased on formation of the antibody-antigen complex on the surface of the electrode. Then increased electrochemical response is thought to result from a combination of beneficial effects including the biocompatibility and large surface area of the AuNPs, the high conductivity of the graphene paste electrode, the synergistic effects of composite film, and the increased quantity of HBsAb adsorbed on the electrode surface. The differential pulse voltammetric responses of the hexacyanoferrate redox pair are proportional to the concentration of HBsAg in the range from 0.5–800?ng?mL^?1, and the detection limit is 0.1?ng?mL^?1 (at an S/N of 3). The immunosensor is sensitive and stable.

Method for determination of microcystin-leucine-arginine in water samples based on the quenching of the fluorescence of bioconjugates between CdSe/CdS quantum dots and microcystin-leucine-arginine antibody

We have developed a method for the determination of microcystin-leucine-arginine (MC-LR) in water samples that is based on the quenching of the fluorescence of bioconjugates between CdSe/CdS quantum dots (QDs) and the respective antibody after binding of MC-LR. The core-shell CdSe/CdS QDs were modified with 2-mercaptoacetic acid to improve water solubility while their high quantum yields were preserved. Monoclonal MC-LR antibody was then covalently bioconjugated to the QDs. It was found that the fluorescence intensity of the bioconjugates was quenched in the presence of MC-LR. A linear relationship exists between the extent of quenching and the concentration of MC-LR. Parameters affecting the quenching were investigated and optimized. The limit of detection is 6.9?×?10^?11 mol L^?1 (3σ). The method was successfully applied to the determination of MC-LR in water samples.

Nanosensor for dopamine and glutathione based on the quenching and recovery of the fluorescence of silica-coated quantum dots

We have constructed a fluorescent nanosensor for dopamine (DA) and glutathione (GSH) in physiologically relevant concentrations. CdTe quantum dots (QDs) were coated with silica, and dopamine-quinone (formed by oxidation of DA) is captured on the surface of silica via dual interactions (hydrogen bonding and electrostatic interaction) and quenches the photoluminescence of the modified QDs by an electron transfer process. GSH, in being a strong reducing agent, can chemically reduce the dopamine-quinone on the QDs, and this results in recovered photoluminescence. There are linear relationships between the concentrations of dopamine and GSH respectively and the intensity of the photoluminescence intensity of the QDs both in the quenched and regenerated form, the ranges being 0.0005 to 0.1 mmol?L^?1 for dopamine, and 0.1 to 10 mmol?L^?1 for GSH. The method was applied to the determination of dopamine and GSH in human serum samples with satisfactory results.

Preparation of liposomes loaded with quantum dots, fluorescence resonance energy transfer studies, and near-infrared in-vivo imaging of mouse tissue

We report on a simple, fast and convenient method to engineer lipid vesicles loaded with quantum dots (QDs) by incorporating QDs into a vesicle-type of lipid bilayer using a phase transfer reagent. Hydrophilic CdTe QDs and near-infrared (NIR) QDs of type CdHgTe were incorporated into liposomes by transferring the QDs from an aqueous solution into chloroform by addition of a surfactant. The QD-loaded liposomes display bright fluorescence, and the incorporation of the QDs into the lipid bilayer leads to enhanced storage stability and reduced sensitivity to UV irradiation. The liposomes containing the QD were applied to label living cells and to image mouse tissue in-vivo using a confocal laser scanning microscope, while NIR images of mouse tissue were acquired with an NIR fluorescence imaging system. We also report on the fluorescence resonance energy transfer (FRET) that occurs between the CdTe QDs (the donor) and the CdHgTe QDs (the acceptor), both contained in liposomes. Based on these data, this NIR FRET system shows promise as a tool that may be used to study the release of drug-loaded liposomes and their in vivo distribution.

Detection of Helicobacter pylori with a nanobiosensor based on fluorescence resonance energy transfer using CdTe quantum dots

We report on a method for the sensitive determination of Helicobacter that is based on fluorescence resonance energy transfer using two oligonucleotide probes labeled with CdTe quantum dots (QDs) and 5-carboxytetramethylrhodamine (Tamra) respectively. QDs labeled with an amino-modified first oligonucleotide, and a Tamra-labeled second oligonucleotide were added to the DNA targets upon which hybridization occurred. The resulting assembly brings the Tamra fluorophore (the acceptor) and the QDs (the donor) into close proximity and causes fluorescence resonance energy transfer (FRET) to occur upon photoexcitation of the donor. In the absence of target DNA, on the other hand, the probes are not ligated, and no emission by the Tamra fluorophore is produced due to the lack of FRET. The feasibility of the method was demonstrated by the detection of a synthetic 210-mer nucleotide derived from Helicobacter on a nanomolar level. This homogeneous DNA detection scheme is simple, rapid and efficient, does not require excessive washing and separation steps, and is likely to be useful for the construction of a nanobiosensor for Helicobacter species.