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.

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.

Fluorescence energy transfer-based multiplexed hybridization assay using gold nanoparticles and quantum dot conjugates on photonic crystal beads

A multiplexed assay strategy was developed for the detection of nucleic acid hybridization. It is based on fluorescence resonance energy transfer (FRET) between gold nanoparticles (AuNPs) and multi-sized quantum dots (QDs) deposited on the surface of silica photonic crystal beads (SPCBs). The SPCBs were first coated with a three-layer primer film formed by the alternating adsorption of poly(allylamine hydrochloride) and poly(sodium 4-styrensulfonate). Probe DNA sequences were then covalently attached to the carboxy groups at the surface of the QD-coated SPCBs. On addition of DNA-AuNPs and hybridization, the fluorescence of the donor QDs is quenched because of the close proximity of the AuNPs. However, the addition of target DNA causes a recovery of the fluorescence of the QD-coated SPCBs, thus enabling the quantitative assay of hybridized DNA. Compared to fluorescent dyes acting as acceptors, the use of AuNPs results in much higher quenching efficiency. The multiplexed assay displays a wide linear range, high sensitivity, and very little cross-reactivity. This work, where such SPCBs are used for the first time in a FRET assay, is deemed to present a new and viable approach towards high-throughput multiplexed gene assays.

Use of quantum dots in the development of assays for cancer biomarkers

Biomarker assays may be useful for screening and diagnosis of cancer if a set of molecular markers can be quantified and statistically differentiated between cancerous cells and healthy cells. Markers of disease are often present at very low concentrations, so methods capable of low detection limits are required. Quantum dots (QDs) are nanoparticles that are emerging as promising probes for ultrasensitive detection of cancer biomarkers. QDs attached to antibodies, aptamers, oligonucleotides, or peptides can be used to target cancer markers. Their fluorescent properties have enabled QDs to be used as labels for in-vitro assays to quantify biomarkers, and they have been investigated as in-vivo imaging agents. QDs can be used as donors in assays involving fluorescence resonance energy transfer (FRET), or as acceptors in bioluminescence resonance energy transfer (BRET). The nanoparticles are also capable of electrochemical detection and are potentially useful for “lab-on-a-chip” applications. Recent developments in silicon QDs, non-blinking QDs, and QDs with reduced-size and controlled-valence further make these QDs bioanalytically attractive because of their low toxicity, biocompatibility, high quantum yields, and diverse surface modification flexibility. The potential of multiplexed sensing using QDs with different wavelengths of emission is promising for simultaneous detection of multiple biomarkers of disease.

Recent advances in the use of near-infrared quantum dots as optical probes for bioanalytical,imaging and solar cell application

Near-infrared quantum dots (NIR QDs) represent a powerful material and diagnostic tool owing to their long emission wavelength which extends into the near-infrared region where permeation depths are much larger and where the intrinsic absorbance and autofluorescence of tissue is much smaller compared to shortwave emitting QDs. We are reviewing here recent (2008–2013) methods for the preparation of NIR QDs, their (bio)chemical modifications, and their applications. The article is subdivided into the following sections: (a) Synthesis of NIR QDs; (b) modification of NIR QDs and probe preparation; (c) applications of NIR QDs (with subsections on fluorescence quenching and fluorescence enhancement-based bioanalytical detection, on fluorescence bioimaging, on uses in photovoltaic cells and solar cells, and on molecular detection based on electrogenerated chemiluminescence). We finally make conclusions and discuss current challenges, trends, and future applications. The review contains 119 references. Figure

This review systematically presents the development, preparation methods, modifications and bioapplications of Near-infrared quantum dots (NIR QDs). The review contains 126 references.     

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.

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.

Quantum dots and polymer hybrid composites: new insights into fluorescence switch and turn-on anion sensing

We present a previously unexplored mechanism for a quantum dot (QD) fluorescence switch, and introduce its application to turn-on sensing of anions. A hybrid composite is formed from polyacrylic acid (PAA) and the surface of CdTe QDs through strong coordination interactions between the carboxy groups of PAA and the Cd atoms on the surface of the QDs. Such interactions cause almost complete quenching of the fluorescence of the QDs via the pull effect of PAA. Carbonate, silicate or phosphate anions are then added in the pH 8.2 solution of the QD-PAA composites. These anions are partially hydrolyzed and protonated, and then react with the coordinating oxygen atoms due to the formation of hydrogen bonds. This leads to a decrease in the pull effect of PAA, and eventually gives rise to a strong recovery of fluorescence. The detection limits are 0.29, 0.02, and 0.76 mM, respectively, for carbonate, silicate and phosphate. The surface modulation method presented here provides a novel strategy for the design of QD-based chemosensors for carbonate, silicate or phosphate which otherwise are difficult to detect.

Atomic force microscopy of model lipid membranes

Supported lipid bilayers (SLBs) are biomimetic model systems that are now widely used to address the biophysical and biochemical properties of biological membranes. Two main methods are usually employed to form SLBs: the transfer of two successive monolayers by Langmuir–Blodgett or Langmuir–Schaefer techniques, and the fusion of preformed lipid vesicles. The transfer of lipid films on flat solid substrates offers the possibility to apply a wide range of surface analytical techniques that are very sensitive. Among them, atomic force microscopy (AFM) has opened new opportunities for determining the nanoscale organization of SLBs under physiological conditions. In this review, we first focus on the different protocols generally employed to prepare SLBs. Then, we describe AFM studies on the nanoscale lateral organization and mechanical properties of SLBs. Lastly, we survey recent developments in the AFM monitoring of bilayer alteration, remodeling, or digestion, by incubation with exogenous agents such as drugs, proteins, peptides, and nanoparticles.

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%.

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.

Aptasensor for adenosine triphosphate based on electrode–supported lipid bilayer membrane

We have developed an aptasensor for adenosine triphosphate (ATP) based on an electrode-supported lipid bilayer membrane. The assay is based on a conformational change that is induced after binding the target which modulates the electron transfer rate in the conductive path. The method is highly sensitive, stable, and repeatable. The detection limit for ATP is 50 nM, and the dynamic range extends to 3.2 μM, which covers the concentration range of ATP in cell lysates (from 0.1 to 1 μM). The method also holds promise in that it may be transferred to submicro- or nano-scale electrodes so to enable intracellular monitoring of ATP.

Surface plasmon resonance, fluorescence, and circular dichroism studies for the characterization of the binding of BACE-1 inhibitors

The mechanism of action underlying β-secretase 1 (BACE-1) inhibition was characterized by a surface plasmon resonance (SPR) method using primary amino groups to immobilize OM99-2, a well-known highly potent peptidic BACE-1 inhibitor, on the carboxyl groups of the dextran layer of a sensor chip. The diluted BACE-1 was mixed with buffer or the test compound and the mixture was flushed through the chip. BACE-1 binding to the immobilized peptide inhibitor was quantified. This SPR method was used to identify BACE-1 inhibitor binding sites and the mechanism of action (competitive/noncompetitive) and to validate findings of fluorescence resonance energy transfer (FRET) inhibition studies. To support this, a multimethodological approach (circular dichroism and fluorescence spectroscopy) was applied in parallel to FRET inhibition studies to characterize the binding modes of peptidic and nonpeptidic BACE-1 inhibitors. Circular dichroism spectroscopy served to correlate the conformation of BACE-1 with enzymatic activity and to monitor secondary structure changes upon ligand binding. In a complementary approach, direct fluorescence spectroscopy was used to characterize different BACE-1 inhibitor binding sites. The influence of pH and inhibitors on BACE-1 secondary structure was also elucidated. This multimethodological approach was applied to identify binding modes of bis(7)-tacrine and myricetin in comparison with well-known peptidic inhibitors.

Fluorometric determination of microRNA via FRET between silver nanoclusters and CdTe quantum dots

This paper describes a CdTe quantum dot-based fluorescence resonance energy transfer (FRET) based assay for the detection of the breast cancer biomarker microRNA. The method relies on energy transfer between DNA-templated silver nanoclusters (AgNCs) and CdTe QDs. Interaction between double strand oligonucleotide and QDs can be detected qualitatively through gel analysis and quantitatively by the signal amplification from AgNCs to QDs via FRET, best measured at an excitation wavelength of 350 nm and at emission wavelengths of 550 and 590 nm. Three microRNAs (microRNA-21, microRNA-155 and Let-7a) were quantified to verify the feasibility of the method, and a high sensitivity for microRNAs was achieved. Fluorescence intensity increases linearly with the log of the concentration of microRNA 155 in the 5.0 pM to 50 nM range, with a 1.2 pM detection limit.

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Graphical abstract Schematic presentation of a quantum dot-based (QD-based) fluorescence resonance energy transfer technique for the detection of microRNA (miRNA). The method relies on energy transfer between DNA-templated silver nanoclusters (AgNCs) and QDs.

     

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.

Ratiometric sensing of mercury(II) based on a FRET process on silica core-shell nanoparticles acting as vehicles

We report on a fluorescence resonance energy transfer (FRET)-based ratiometric sensor for the detection of Hg(II) ion. First, silica nanoparticles were labeled with a hydrophobic fluorescent nitrobenzoxadiazolyl dye which acts as a FRET donor. A spirolactam rhodamine was then covalently linked to the surface of the silica particles. Exposure of the nanoparticles to Hg(II) in water induced a ring-opening reaction of the spirolactam rhodamine moieties, leading to the formation of a fluorescent derivative that can serve as the FRET acceptor. Ratiometric sensing of Hg(II) was accomplished by ratioing the fluorescence intensities at 520 nm and 578 nm. The average decay time for the donor decreases from 9.09 ns to 7.37 ns upon addition of Hg(II), which proves the occurrence of a FRET process. The detection limit of the assay is 100 nM (ca. 20 ppb). The sensor also exhibits a large Stokes shift (>150 nm) which can eliminate backscattering effects of excitation light.

Fluorescent turn-on detection of cysteine using a molecularly imprinted polyacrylate linked to allylthiol-capped CdTe quantum dots

CdTe quantum dots capped with thioglycolic acid (TGA) display a strong turn-on fluorescence response if exposed to solutions of cysteine (Cys). In order to exploit this effect, a molecularly imprinted polymer (MIP) for Cys was covalently linked to the QDs via allyl mercaptan. The resulting nanomaterials (QDs, MIP-coated QDs, and nonimprint-coated QDs) were characterized by FTIR and scanning electron microscopy. The adsorption of Cys was studied in phosphate buffer (pH 7.4) with respect to equilibration times (5, 15, and 40 min, respectively), binding constants [2.98, 2.42, and 0.96 (×10⁴ M^?1)], and Langmuir isotherms (R²?=?0.9995, 0.9999, and 0.9983) in the Cys concentration range between 3.33 μM to 500 μM. The method has a detection limit of 0.85 μM (3σ, blank, for n?=?10). The selectivity of the MIP-coated QDs for Cys over 19 other amino acids is similar to that of bare QDs, but MIP-QDs afford better recoveries of Cys from solutions also containing bovine serum albumin (90 %) and fetal bovine serum (97 %), respectively, when compared to the recoveries that are obtained with bare (non-imprinted) QDs (135 % and 120 %). This is probably due to the fact that the outer MIP shell largely reduces protein wrapping, dot aggregation, and matrix inclusion.

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.

Electrochemiluminescence immunoassay at a nanoporous gold leaf electrode and using CdTe quantun dots as labels

An electrochemiluminescence-based immunoassay using quantum dots (QDs) as labels for the carcinoembryonic antigen (CEA) was developed using an electrode modified with leafs of nanoporous gold. CEA was initially immobilized on the electrode via a sandwich immunoreaction, and then CdTe quantum dots capped with thioglycolic acid were used to label the second antibody. The intensity of the ECL of the QDs reflects the quantity of CEA immobilized on the electrode. Thus, in the presence of dithiopersulfate as the coreactant, the ECL serves as the signal for the determination of CEA. The intensity of the electroluminescence (ECL) of the electrode was about 5.5-fold higher than that obtained with a bare gold electrode. The relation between ECL intensity and CEA concentration is linear in the range from 0.05 to 200?ng.mL^-1, and the detection limit is 0.01?ng.mL^-1. The method has the advantages of high sensitivity, good reproducibility and long-term stability, and paves a new avenue for applying quantum dots in ECL-based bioassays.

In vitro and in vivo imaging with quantum dots

Quantum dots (QDs), also named semiconductor nanocrystals, have initiated a new realm of bioscience by combining nanomaterials with biology, which will profoundly influence future biological and biomedical research. In this review, we describe the extraordinary optical properties of QDs and developments in methods for their synthesis. We focus on fluorescent imaging with QDs both in vitro and in vivo, and the cytotoxicity of QDs and potential barriers to their use in practical biomedical applications. Finally, we provide insights into improvements aimed at decreasing the cytotoxicity of QDs and the future outlook of QD applications in biomedical fields.