Unconventional Application of Conventional Enzymatic Substrate: First Fluorogenic Immunoassay Based on Enzymatic Formation of Quantum Dots

In this study, a simple fluorogenic immunoassay based on in situ formation of semiconductor quantum dots (QDs) is described. We discovered that alkaline phosphatase (ALP), the enzyme broadly used in enzyme-linked immuno-sorbent assay (ELISA), is able to trigger formation of fluorescent CdS QDs. ALP-catalyzed hydrolysis of <i>p</i>-nitrophenyl phosphate (pNPP) leads to the formation of <i>p</i>-nitrophenol and inorganic phosphate. The latter stabilizes CdS QDs produced in situ through interaction of Cd²⁺ with S^2– ions. So, the specific interaction of analyte (antibody) with ALP-labeled antibody can be detected through formation of CdS QDs, monitored by recording emission spectra at λ_ex = 290 nm. The fluorescence intensity showed to be dependent on the concentration of target antibody. This method allowed us to detect as low as 0.4 ng mL^–1 of analyte antibody with a linear range up to 10 ng mL^–1. The sensitivity of this novel assay showed to be 1 order of magnitude better than that of the standard method based on colorimetric <i>p</i>-nitrophenyl phosphate assay

Enzymatic Product-Mediated Stabilization of CdS Quantum Dots Produced <i>In Situ</i>: Application for Detection of Reduced Glutathione, NADPH, and Glutathione Reductase Activity

Glutathione is the most abundant nonprotein molecule in the cell and plays an important role in many biological processes, including the maintenance of intracellular redox states, detoxification, and metabolism. Furthermore, glutathione levels have been linked to several human diseases, such as AIDS, Alzheimer disease, alcoholic liver disease, cardiovascular disease, diabetes mellitus, and cancer. A novel concept in bioanalysis is introduced and applied to the highly sensitive and inexpensive detection of reduced glutathione (GSH), over its oxidized form (GSSG), and glutathione reductase (GR) in human serum. This new fluorogenic bioanalytical system is based on the GSH-mediated stabilization of growing CdS nanoparticles. The sensitivity of this new assay is 5 pM of GR, which is 3 orders of magnitude better than other fluorogenic methods previously reported

Assays for Methionine γ‑Lyase and <i>S</i>‑Adenosyl‑l‑homocysteine Hydrolase Based on Enzymatic Formation of CdS Quantum Dots <i>in Situ</i>

<i>S</i>-Adenosyl-l-homocysteine hydrolase (AHCY) hydrolyzes its substrate <i>S</i>-adenosyl-l-homocysteine (AdoHcy) to l-homocysteine (Hcy). Methionine γ-lyase (MGL) catalyzes the decomposition of Hcy to hydrogen sulfide which forms fluorescent CdS nanoparticles in the presence of Cd­(NO₃)₂. On the basis of these enzymatic reactions, two new simple and robust fluorogenic enzymatic assays for MGL and AHCY were developed and applied to detection of AHCY inhibitors

Microbead QD-ELISA: Microbead ELISA Using Biocatalytic Formation of Quantum Dots for Ultra High Sensitive Optical and Electrochemical Detection

Electrochemical detection strategies employing semiconductor quantum dots (QDs) open up new opportunities for highly sensitive detection of biological targets. We designed a new assay based on microbead linked enzymatic generation of CdS QDs (Microbead QD-ELISA) and employed it in optical and electrochemical affinity assays for the cancer biomarker superoxide dismutase 2 (SOD2). Biotinylated antibodies against SOD2 were immobilized on the surface of polyvinyl chloride microbeads bearing streptavidin. In order to prevent any non-specific adsorption the microbeads were further blocked with bovine serum albumin. The analyte, SOD2 was captured on microbeads and labeled with alkaline phosphatase-conjugated antibody linked with mouse antibody against SOD2. Hydrolysis of <i>para</i>-nitrophenylphosphate by immobilized alkaline phosphatase triggered the rapid formation of phosphate-stabilized CdS QDs on the surface of microbeads. The resulting semiconductor nanoparticles were detected by fluorescence spectroscopy, microscopy, and square-wave voltammetry (SWV). The electrochemical assay based on the detection with square-wave voltammograms of Cd²⁺ ions originating from immobilized CdS QDs showed linearity up to 45 ng mL^–1, and the limit of SOD2 detection equal to 0.44 ng mL^–1 (1.96 × 10^–11 M). This detection limit is lower by 2 orders of magnitude in comparison with that of other previously published assays for superoxide dismutase. The electrochemical assay was validated with HepG2 (Human hepatocellular carcinoma) cell lysate containing SOD2

Peroxidase-Mimicking DNAzyme Modulated Growth of CdS Nanocrystalline Structures in Situ through Redox Reaction: Application to Development of Genosensors and Aptasensors

This work demonstrates the use of the peroxidase-mimicking DNAzyme (peroxidase-DNAzyme) as general and inexpensive platform for development of fluorogenic assays that do not require organic fluorophores. The system is based on the affinity interaction between the peroxidase-DNAzyme bearing hairpin sequence and the analyte (DNA or low molecular weight molecule), which changes the folding of the hairpin structure and consequently the activity of peroxidase-DNAzyme. Hence, in the presence of the analyte the peroxidase-DNAzyme structure is disrupted and does not catalyze the aerobic oxidation of l-cysteine to cystine. Thus, l-cysteine is not removed from the system and the fluorescence of the assay increases due to the in situ formation of fluorescent CdS nanocrystals. The capability of the system as a platform for fluorogenic assays was demonstrated through designing model geno- and aptasensor for the detection of a tumor marker DNA and a low molecular weight analyte, adenosine 5′triphosphate (ATP), respectively

Blocked Enzymatic Etching of Gold Nanorods: Application to Colorimetric Detection of Acetylcholinesterase Activity and Its Inhibitors

The anisotropic morphology of gold nanorods (AuNRs) has been shown to lead to nonuniform ligand distribution and preferential etching through their tips. We have recently demonstrated that this effect can be achieved by biocatalytic oxidation with hydrogen peroxide, catalyzed by the enzyme horseradish peroxidase (HRP). We report here that modification of AuNRs with thiol-containing organic molecules such as glutathione and thiocholine hinders enzymatic AuNR etching. Higher concentrations of thiol-containing molecules in the reaction mixture gradually decrease the rate of enzymatic etching, which can be monitored by UV–vis spectroscopy through changes in the AuNR longitudinal plasmon band. This effect can be applied to develop novel optical assays for acetylcholinesterase (AChE) activity. The biocatalytic hydrolysis of acetylthiocholine by AChE yields thiocholine, which prevents enzymatic AuNR etching in the presence of HRP. Additionally, the same bioassay can be used for the detection of nanomolar concentrations of AChE inhibitors such as paraoxon and galanthamine

Aminoferrocene-Based Prodrugs and Their Effects on Human Normal and Cancer Cells as Well as Bacterial Cells

Aminoferrocene-based prodrugs are activated under cancer-specific conditions (high concentration of reactive oxygen species, ROS) with the formation of glutathione scavengers (<i>p</i>-quinone methide) and ROS-generating iron complexes. Herein, we explored three structural modifications of these prodrugs in an attempt to improve their properties: (a) the attachment of a −COOH function to the ferrocene fragment leads to the improvement of water solubility and reactivity in vitro but also decreases cell-membrane permeability and biological activity, (b) the alkylation of the <i>N</i>-benzyl residue does not show any significant affect, and (c) the attachment of the second arylboronic acid fragment improves the toxicity (IC₅₀) of the prodrugs toward human promyelocytic leukemia cells (HL-60) from 52 to 12 μM. Finally, we demonstrated that the prodrugs are active against primary chronic lymphocytic leukemia (CLL) cells, with the best compounds exhibiting an IC₅₀ value of 1.5 μM. The most active compounds were found to not affect mononuclear cells and representative bacterial cells