Molecular-Counting-Free and Electrochemiluminescent Single Molecular Immunoassay with Dual-stabilizers-capped CdSe Nanocrystals as Labels (original) (raw)
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Analytical Chemistry, 2016
Biorelated single-molecule detection (SMD) has been achieved typically by imaging the redox fluorescent labels and then determining each label one by one. Herein, we demonstrated that the capping agents (i.e., mercaptopropionic acid and sodium hexametaphosphate) can facilitate the electrochemical involved hole (or electron) injecting process and improve the stability of the dual-stabilizers-capped CdSe nanocrystals (NCs), so that the CdSe NCs could be electrochemically and repeatedly inspired to excited states by giving off electrochemiluminescence (ECL) in a cyclic pattern. With the CdSe NCs as ECL label and carcinoembryonic antigen (CEA) as target molecule, a convenient single-molecule immunoassay was proposed by simply detecting the ECL intensity of the dual-stabilizers-capped CdSe NCs in a sandwich-typed immune complex. The limit of detection is 0.10 fg/mL at S/N = 3, which corresponds to about 6−8 CEA molecules in 20 μL of serum sample. Importantly, the ECL spectra of both CdSe NCs and its conjugate with probe antigen in the immune complex were almost identical to the photoluminescence spectrum of bare CdSe NCs, indicating that all emissions were originated from the same excited species. The molecular-counting-free and ECL-based SMD might be a promising alternative to the fluorescent SMD.
Analytical chemistry, 2017
A spectrum-resolved dual-color electrochemiluminescence (ECL) immunoassay was designed and implemented to simultaneously detect carcinoembryonic antigen (CEA) and alpha fetoprotein (AFP) with CdTe (λmax = 776 nm) and CdSe (λmax = 550 nm) nanocrystals (NCs) as ECL tags. The CdTe and CdSe NCs were labeled with respective probe antibodies (Ab2) of CEA and AFP, respectively, and then immobilized onto the working electrode surface via sandwich-type immunoreactions. Both CdTe and CdSe NCs within the NCs immunocomplexes can be electrochemically reduced and simultaneously give off monochromatic ECL emissions in the near-infrared and greenish regions, respectively, when (NH4)2S2O8 was used as a cathodic ECL coreactant. The ECL spectra of the two surface-confined NCs were well separated and had no cross energy-transfer interactions, which made the dual-color immunoassay highly selective and sensitive toward respective target analytes. With the proposed ECL biosensor, CEA and AFP were simultan...
Spectrum-Based Electrochemiluminescent Immunoassay with Ternary CdZnSe Nanocrystals as Labels
Analytical Chemistry, 2016
Conventional electrochemiluminescence (ECL) research has been performed by detecting the total photons (i.e., the ECL intensity). Herein, systematic spectral exploration on the ECL of dual-stabilizers-capped ternary CdZnSe nanocrystals (NCs) and its sensing application were carried out on a homemade ECL spectral acquiring system. The ternary CdZnSe NCs could be repeatedly injected with electrons via some electrochemical ways and then result in strong cathodic ECL with the coupling of ammonium persulfate. ECL spectrum of the CdZnSe NCs was almost identical to corresponding photoluminescence spectrum, indicating that the excited states of CdZnSe NCs in ECL were essentially the same as those in photoluminescence. Importantly, after being labeled to the probe antibody (Ab 2) of α-fetal protein (AFP) antigen, the ternary NCs in the Ab 2 |NCs conjugates could preserve their ECL spectrum very well. A spectrum-based ECL immunoassay was consequently proposed with the CdZnSe NCs as ECL tags and AFP as target molecules. The limit of detection is 0.010 pg/mL, with a signal-to-noise (S/N) ratio of 3, indicating a sensitive ECL sensing strategy that was different from the conventional ones. This work might open a pathway to the spectrally resolved ECL analysis with even-higher S/N ratios than the fluorescent analysis.
The electrochemical behavior of a trinuclear ruthenium(II)containing complex, [((phen) 2 Ru(dpp)) 2 RhCl 2 ] 5+ (where phen) 1,10-phenanthroline, dpp) 2,3-bis(2-pyridyl)pyrazine), was studied in acetonitrile (MeCN) and aqueous solutions. In MeCN containing 0.10 M tetran-butylammonium perchlorate (TBAP), the complex displayed a reversible, overlapping Ru II/III redox process with E 1/2) +1.21 V vs Ag/Ag + (10 mM), an irreversible reduction of Rh III/I at-0.73 V vs Ag/Ag + , and two quasi-reversible dpp/dppcouples with E 1/2)-1.11 and-1.36 V vs Ag/Ag + at a Pt electrode with a scan rate of 50 mV s-1. In 0.20 M Tris buffer solution (pH 7.4), an irreversible, overlapping Ru II/III oxidation at +1.48 V vs Ag/AgCl (3 M KCl), and an irreversible reduction of Rh III/II at-0.78 V vs Ag/AgCl were observed at a glassy carbon electrode with a scan rate of 50 mV/s. Investigations on the electrogenerated chemiluminescence (ECL) of the complex revealed that 2-(dibutylamino) ethanol (DBAE) was superior to trin-propylamine (TPrA) as an ECL coreactant within their entire concentration range of 10-100 mM in MeCN, and in aqueous media, as low as 1.0 nM of the complex can be detected using TPrA coreactant ECL. A maximum ECL emission of 640 nm, which is about 55 nm blue shift to its fluorescence, was observed in MeCN with DBAE as a coreactant. Interactions of the complex with calf thymus DNA (ctDNA) were conducted with a flow-cell based quartz-crystal microbalance, and a binding constant of 2.5 × 10 5 M-1 was calculated on the basis of the Langmuir isotherm equation. Electrogenerated chemiluminescence (ECL), a process of light generation initiated by electrochemical reactions, has proven to be a very powerful analytical technique and been widely used in the areas of, for example, DNA probe, immunoassay, biowarfare agent testing, and environmental detection, because of its inherent
Application of Antibody-Conjugated CdSe/MSA Quantum Dots on Immunohistochemistry
SSR Institute of International Journal of Life Sciences, 2020
Background: Quantum dots (QDs) are recently conjugated to antibody for using in biological labeling applications. In previous studies, we developed CdSe/MSA QDs, which were coated with protein A/G (pA/G) for antibody conjugation, and evaluated their cell staining application. Here, we expanded their applications into immunohistochemistry (IHC) by investigating their storage time by accelerated heat aging method, and comparing them with FITC in sample labeling. Methods: Cytokeratin 6A (KRT6A) in animal skin tissue section were stained by pA/G-coated QDs conjugated to anti-KRT6A antibody and TF-1 human erythroleukemia cells were stained by pA/G-coated QDs conjugated to anti-CD34 antibody. Results: The results indicated that our pA/G-coated QDs effectively stained KRT6A in epidermis of skin tissue section when they were probed with specific antibody. Besides, pA/G-coated QDs still maintained their labeling ability in cell staining and IHC formats after 12-month storage at 4 o C. In comparison with FITC (relatively almost the same emission fluorophore), our QDs showed a significantly stronger fluorescent signal. Conclusion: In conclusion, these results indicated that our pA/G-coated QDs were potentially fluorescent nanomaterials and could be applied in a wide range of biological labeling applications.
Single-domain antibody functionalized CdSe/ZnS quantum dots for cellular imaging of cancer cells
The Journal of Physical …, 2008
Synthesis and characterization of CdSe Core QDs and CdSe/ZnS Core/Shell QDs. TOP, TOPO or TOPO/HDA-capped CdSe nanocrystals were synthesized using published methods with modification. i For a typical reaction, the mixture of 2.5 mmol of CdO, 7.0 mmol of stearic acid, and 50 ml of ODE in a 250 mL three-neck flask was heated to about 200 ~ 290 °C to obtain a clear colorless solution. After this solution was cooled to 100 ~ 120 °C temperature, HDA (18 g) and of TOPO (10 g) were added into the flask. Under nitrogen flow, this system was reheated to 280 °C. At this temperature, 2.1 ml of Se-TBP solution was quickly injected, the solution of which was made by dissolving 20 mmol of Se in 10 ml of TBP. The growth temperature was then reduced to 260 ~ 280 °C for 10 ~ 15 min. The reaction mixture
Biosensors and Bioelectronics, 2019
An ultrahigh sensitive, simple and reliable Electrochemiluminescence (ECL) immunosensor for selective quantification of p53 protein was designed according to the enhancement effects of AuNPs on ECL emission of CdS nanocrystals (CdS NCs). CdS NCs were immobilized on the glassy carbon electrode and AuNPs introduced to the process through formation of a sandwichtype immunocomplex between first anti-p53/p53/ secondary anti-p53. ECL of CdS NCs firstly evoked the SPR of AuNPs which in return amplified the CdS NCs ECL intensity. By using graphene oxide in immunosensor fabrication procedure, and attaching more AuNPs on the 2 surface of the electrode, the ECL intensity was further increased resulting in much higher sensitivity. After applying the optimum conditions, the linear range of the developed immunosensor was found between 20 to 1000 fg/ml with a calculated limit of detection of 4 fg/ml. Moreover, the interference, reproducibility and storage stability studies of the immunosensor were investigated. Finally, immunosensor's authenticity was evaluated by detecting the p53 protein in human spikes which offers it as a potential in early detection of cancer, monitoring the cancer progress and clinical prognosis.
Synthesis of mercaptopropionic acid stabilized CDS quantum dots for bioimaging in breast cancer
Semiconductor inorganic nanocrystals or Quantum dots (QDs) are nowadays extensively used for imaging and analysis of biomolecules owing to their superior optical properties over conventional organic fluorophores. They have excellent potential for synthesizing molecular probes against various biological markers such as free antigens, cell surface markers/antigens, bacteria, viruses and tissues. Traditional synthesis protocols of the QDs generally lead to the formation of hydrophobic nanocrystals. For biological applications, post-synthesis modifications need to be introduced to render required hydrophilicity.However, such additional steps make the tiny QDs structures bulky, which is unwanted in subsequent in-vivo executions. The present work reportsa simple method for the direct synthesis of hydrophilic carboxyl (-COOH) functionalized CdS QDs using mercaptopropionic acid as a sulfur source and stabilizer. This aqueous synthesis route avoidsthe requirement of extra surface modification steps.
Simultaneous electrochemical immunoassay using CdS/DNA and PbS/DNA nanochains as labels
Biosen. Bioelectron., 2013
An electrochemical method for the simultaneous detection of two different tumor markers, carcinoembryonic antigen (CEA) and a-fetoprotein (AFP), in one-pot, using CdS/DNA and PbS/DNA nanochains as labels was developed. Herein, magnetic beads (MBs) as bimolecule immobilizing carriers, were used for co-immobilization of primary anti-CEA and anti-AFP antibodies. The distinguishable signal labels were synthesized by in situ growth of CdS and PbS nanoparticles on DNA chains, respectively, which were further employed to label the corresponding secondary antibodies. A sandwich-type immunoassay format was formed by the biorecognition of the antigens and corresponding antibodies. The assay was based on the peak currents of Cd 2 þ and Pb 2 þ dissolved from CdS and PbS nanoparticles by HNO 3 using square wave stripping voltammetry. Experimental results show that the multiplexed electrochemical immunoassay has enabled the simultaneous monitoring of CEA and AFP in a single run with wide working ranges of 0.1-100 ng mL À 1 for CEA and 0.5-200 ng mL À 1 for AFP. The detection limits reach to 3.3 pg mL À 1 for CEA and 7.8 pg mL À 1 for AFP.