Seonhwa Park

A BODIPY-Based Probe for the Selective Detection of Hypochlorous Acid in Living Cells

Reactive oxygen species (ROS) are essential for a wide range of biological and pathological events. [1] During infection and inflammation, the phagocytic leukocytes, including neutrophils, monocytes, and macrophages generate reactive oxygen species (ROS) to kill invading bacteria and pathogens. [2] Among ROS, hypochlorous acid (HOCl/OCl )i s a highly reactive oxygen species produced from hydrogen peroxide (H2O2) and chloride ions (Cl ) by the enzyme myeloperoxidase (MPO), which is secreted by activated neutrophils. [3] Although hypochlorous acid plays important roles in the human immune-defense system, overproduction of ROS in living organism has detrimental effects on biological molecules, including nucleic acids, lipids, and proteins, resulting in the inhibition of various protein functions, and contributes to the progression of numerous human diseases, such as atherosclerosis, cancer, cardiovascular diseases, and rheumatoid arthritis. [4] Despite its importance in human health and disease, not as much is known about the mechanism of action and specific roles of HOCl in living systems in comparison with other ROS, owing to slower progress in the development of suitable probes. Several fluorescence probes for the detection and visualization of HOCl in living cells have recently been developed on the basis of the strong oxidizing properties of HOCl. [5–7] HOCl-induced oxidation reactions were employed in the design of fluorescent probes in which the fluorescence properties were regulated by the conversion of the spirocyclic form of rhodamine fluorophores into their ringopened form, [5] or through photoinduced-electron-transfer processes. [6] To facilitate practical applications of such probes, next-generation designs should emphasize higher analyte selectivity, limit susceptibility to autooxidation, and avoid demanding multistep syntheses. Herein, we report the facile synthesis and properties of a new boron-dipyrromethene (BODIPY) dye bearing a methylthioether group, and its biological application as a highly sensitive and selective

Hydroquinone Diphosphate as a Phosphatase Substrate in Enzymatic Amplification Combined with Electrochemical–Chemical–Chemical Redox Cycling for the Detection of E. coli O157:H7

Signal amplification by enzyme labels in enzyme-linked immunosorbent assays (ELISAs) is not sufficient for detecting a low number of bacterial pathogens. It is useful to employ approaches that involve multiple signal amplification such as enzymatic amplification plus redox cycling. An advantageous combination of an enzyme product [for fast electrochemical-chemical-chemical (ECC) redox cycling that involves the product] and an enzyme substrate (for slow side reactions and ECC redox cycling that involve the substrate) has been developed to obtain a low detection limit for E. coli O157:H7 in an electrochemical ELISA that employs redox cycling. In our search for an alkaline phosphatase substrate/product couple that is better than the most common couple of 4-aminophenyl phosphate (APP)/4-aminophenol (AP), we compared five couples: APP/AP, hydroquinone diphosphate (HQDP)/hydroquinone (HQ), L-ascorbic acid 2-phosphate/L-ascorbic acid, 4-amino-1-naphthyl phosphate/4-amino-1-naphthol, and 1-naphthyl phosphate/1-naphthol. In particular, we examined signal-to-background ratios in ECC redox cycling using Ru(NH(3))(6)(3+) and tris(2-carboxyethyl)phosphine as an oxidant and a reductant, respectively. The ECC redox cycling that involves HQ is faster than the cycling that involves AP, whereas the side reactions and ECC redox cycling that involve HQDP are negligible compared to the APP case. These results seem to be due to the fact that the formal potential of HQ is lower than that of AP and that the formal potential of HQDP is higher than that of APP. Enzymatic amplification plus ECC redox cycling based on a HQDP/HQ couple allows us to detect E. coli O157:H7 in a wide range of concentrations from 10(3) to 10(8) colony-forming units/mL.

Glucose-Oxidase Label-Based Redox Cycling for an Incubation Period-Free Electrochemical Immunosensor

Catalytic reactions of enzyme labels in enzyme-linked immunosorbent assays require a long incubation period to obtain high signal amplification. We present herein a simple immunosensing scheme in which the incubation period is minimized without a large increase in the detection limit. This scheme is based on electrochemical-enzymatic (EN) redox cycling using glucose oxidase (GOx) as an enzyme label, Ru(NH3)6(3+) as a redox mediator, and glucose as an enzyme substrate. Fast electron mediation of Ru(NH3)6(3+) between the electrode and the GOx label attached to the electrode allows high signal amplification. The acquisition of chronocoulometric charges at a potential in the mass transfer-controlled region excludes the influence of the kinetics of Ru(NH3)6(2+) electrooxidation and also facilitates high signal-to-background ratios. The reaction between reduced GOx and Ru(NH3)6(3+) is rapid even in air-saturated Tris buffer, where the faster competitive reaction between reduced GOx and dissolved oxygen also occurs. The direct electrooxidation of glucose at the electrode and the direct electron transfer between glucose and Ru(NH3)6(3+) that undesirably increase background levels occur relatively slowly. The detection limit for the EN redox cycling-based detection of cancer antigen 125 (CA-125) in human serum is slightly higher than 0.1 U/mL for the incubation period of 0 min, and the detection limits for the incubation periods of 5 and 10 min are slightly lower than 0.1 U/mL, indicating that the detection limits are almost similar irrespective of the incubation period and that the immunosensor is highly sensitive.

Electroreduction-Based Electrochemical-Enzymatic Redox Cycling for the Detection of Cancer Antigen 15-3 Using Graphene Oxide-Modified Indium–Tin Oxide Electrodes

We compare herein biosensing performance of two electroreduction-based electrochemical-enzymatic (EN) redox-cycling schemes [the redox cycling combined with simultaneous enzymatic amplification (one-enzyme scheme) and the redox cycling combined with preceding enzymatic amplification (two-enzyme scheme)]. To minimize unwanted side reactions in the two-enzyme scheme, β-galactosidase (Gal) and tyrosinase (Tyr) are selected as an enzyme label and a redox enzyme, respectively, and Tyr is selected as a redox enzyme label in the one-enzyme scheme. The signal amplification in the one-enzyme scheme consists of (i) enzymatic oxidation of catechol into o-benzoquinone by Tyr and (ii) electroreduction-based EN redox cycling of o-benzoquinone. The signal amplification in the two-enzyme scheme consists of (i) enzymatic conversion of phenyl β-d-galactopyranoside into phenol by Gal, (ii) enzymatic oxidation of phenol into catechol by Tyr, and (iii) electroreduction-based EN redox cycling of o-benzoquinone including further enzymatic oxidation of catechol to o-benzoquinone by Tyr. Graphene oxide-modified indium-tin oxide (GO/ITO) electrodes, simply prepared by immersing ITO electrodes in a GO-dispersed aqueous solution, are used to obtain better electrocatalytic activities toward o-benzoquinone reduction than bare ITO electrodes. The detection limits for mouse IgG, measured with GO/ITO electrodes, are lower than when measured with bare ITO electrodes. Importantly, the detection of mouse IgG using the two-enzyme scheme allows lower detection limits than that using the one-enzyme scheme, because the former gives higher signal levels at low target concentrations although the former gives lower signal levels at high concentrations. The detection limit for cancer antigen (CA) 15-3, a biomarker of breast cancer, measured using the two-enzyme scheme and GO/ITO electrodes is ca. 0.1 U/mL, indicating that the immunosensor is highly sensitive.

Gold nanorods for target selective SPECT/CT imaging and photothermal therapy in vivo

The development of theranostic agents with high detection sensitivity and antitumor efficacy at low concentration is a challenging task for target selective imaging and therapy of cancers. In this study, folate-conjugated and radioactive-iodine-labeled gold nanorods (GNRs) were designed and synthesized for target selective SPECT/CT imaging and subsequent thermal ablation of folate-receptor-overexpressing cancers. Both (ortho-pyridyl) disulfide-poly(ethylene glycol)-folate and a short peptide, H(2)N-Tyr-Asn-Asn-Leu-Ala-Cys-OH, were conjugated on the surface of the GNRs through thiol chemistry. The tyrosine in the peptide sequence was introduced for radioactive-iodine labeling through an iodine-tyrosine interaction. The labeling efficiency of radioactive iodine was more than 99%. Radiochemical stability tests on iodine-125-labeled GNRs in human serum showed that 91% of the iodine-125 remained intact on the GNRs after incubation for 24 h. In vitro and in vivo results in this study confirmed the potential utility of folate-conjugated and iodine-125-labeled GNRs as smart theranostic agents. This type of platform may also be useful for the targeted SPECT/CT imaging and photothermal therapy of inflammatory diseases such as atherosclerosis and arthritis, in which folate-receptor-overexpressing macrophages play pivotal roles.

Low-interference washing-free electrochemical immunosensor using glycerol-3-phosphate dehydrogenase as an enzyme label.

In washing-free electrochemical detection, various redox and reactive species cause significant interference. To minimize this interference, we report a washing-free electrochemical immunosensor using flavin adenine dinucleotide (FAD)-dependent glycerol-3-phosphate dehydrogenase (GPDH) and glycerol-3-phosphate (GP) as an enzyme label and its substrate, respectively, because the reaction of FAD-dependent dehydrogenases with dissolved O2 is slow and the level of GP preexisting in blood is low (<0.1 mM). A combination of a low electrocatalytic indium-tin oxide (ITO) electrode and fast electron-mediating Ru(NH3)6(3+) is employed to obtain a high signal-to-background ratio via proximity-dependent electron mediation of Ru(NH3)6(3+) between the ITO electrode and the GPDH label. Electrochemical oxidation of GPDH-generated Ru(NH3)6(2+) is performed at 0.05 V vs Ag/AgCl, at which point the electrochemical interference is very low. When a washing-free immunosensor is applied to cardiac troponin I detection in human serum, the calculated detection limit is approximately 10 pg/mL, indicating that the immunosensor is very sensitive in spite of the use of washing-free detection with a short detection period (10 min for incubation and 100 s for electrochemical measurement). The low-interference washing-free electrochemical immunosensor shows good promise for fast and simple point-of-care testing.

Washing-Free Heterogeneous Immunosensor Using Proximity-Dependent Electron Mediation between an Enzyme Label and an Electrode

Washing processes, essential in most heterogeneous labeled assays, have been a big hurdle in simplifying the detection procedure and reducing assay time. Nevertheless, less attention has been paid to washing-free heterogeneous labeled assays. We report a purely washing-free immunosensor that allows fast, sensitive, and single-step detection of prostate-specific antigen in serum with low interference. Proximity-dependent electron mediation of ferrocenemethanol (Fc) between an indium-tin oxide (ITO) electrode and a glucose-oxidase (GOx) label allows us to discriminate between a bound and an unbound label: a bound label offers faster electron mediation than an unbound one. The electrooxidation of Fc at a low applied potential (0.13 V vs Ag/AgCl) and a low electrocatalytic ITO electrode and the oxidation of l-ascorbic acid by l-ascorbate oxidase minimize the effect of the interfering species. With a high concentration of glucose (200 mM), the signal and background levels are hardly dependent on the glucose-concentration variation in the sample. The washing-free immunosensor can detect a concentration of ca. 1 pg/mL for mouse IgG in phosphate-buffered saline and a concentration of ca. 10 pg/mL for prostate-specific antigen spiked in female serum after an incubation period of 10 min. The concentrations measured with actual clinical serum samples are in good agreement with the concentrations measured with a commercial instrument, which renders the washing-free heterogeneous immunosensor appealing for practical use.

Facile electrochemical detection of botulinum neurotoxin type E using a two-step proteolytic cleavage

Facile electrochemical methods for measuring protease concentration or protease activity are essential for point-of-care testing of toxic proteases. However, electrochemical detection of proteases, such as botulinum neurotoxin type E (BoNT/E), that cleave a peptide bond between two specific amino acid residues is challenging. This study reports a facile and sensitive electrochemical method for BoNT/E detection. The method is based on a two-step proteolytic cleavage using a target BoNT/E light chain (BoNT/E-LC) and an externally supplemented exopeptidase, L-leucine-aminopeptidase (LAP). BoNT/E-LC cleaves a peptide bond between arginine and isoleucine in IDTQNRQIDRI-4-amino-1-naphthol (oligopeptide-AN) to generate isoleucine-AN. Subsequently, LAP cleaves a bond between isoleucine and AN to liberate a free electroactive AN species. The liberated AN participates in electrochemical-chemical-chemical (ECC) redox cycling involving Ru(NH3)6(3+), AN, and a reducing agent, which allows a high signal amplification. Electrochemical detection is carried out without surface modification of indium-tin oxide electrodes. We show that dithiothreitol is beneficial for enhancing the enzymatic activity of BoNT/E-LC and also for achieving a fast ECC redox cycling. An incubation temperature of 37°C and the use of phosphate buffered saline (PBS) buffer resulted in optimal signal-to-background ratios for efficient BoNT/E detection. BoNT/E-LC could be detected at concentrations of approximately 2.0 pg/mL, 0.2, and 3 ng/mL after 4h, 2h, and 15 min incubation in PBS buffer, respectively, and approximately 0.3 ng/mL after 2-h incubation in bottled water. The method developed could be applied in fast, sensitive, and selective detection of any protease that cleaves a peptide bond between two specific amino acid residues.

Ultrasensitive Protease Sensors Using Selective Affinity Binding, Selective Proteolytic Reaction, and Proximity-Dependent Electrochemical Reaction

The development of a fast and ultrasensitive protease detection method is a challenging task. This paper reports ultrasensitive protease sensors exploiting (i) selective affinity binding, (ii) selective proteolytic reaction, and (iii) proximity-dependent electrochemical reaction. The selective affinity binding to capture IgG increases the concentration of the target protease (trypsin as a model protease) near the electrode, and the selective proteolytic reaction by trypsin increases the concentration of the redox-active species near the electrode. The electrochemical reaction, which is more sensitive to the concentration of the redox-active species near the electrode than to its bulk concentration, provides an increased electrochemical signal, which is further amplified by the electrochemical-chemical redox cycling. An indium-tin oxide electrode modified with reduced graphene oxide, avidin, and biotinylated capture IgG is used as the electrode, and p-aminophenol liberated from an oligopeptide is used as the redox-active species. The new sensor scheme using no washing process is compared with the new sensor scheme using washing process, and with the conventional scheme using only proteolytic reaction. The new scheme provides a higher signal-to-background ratio and a lower detection limit. Moreover, the increased electrochemical signal offers a more selective protease detection. Trypsin can be detected in phosphate-buffered saline and in artificial serum containing l-ascorbic acid with a low detection limit of 0.5 pg/mL, over a wide range of concentrations, and with an incubation period of only 30 min without washing process. The washing-free electrochemical protease sensor is highly promising for simple, fast, ultrasensitive, and selective point-of-care testing of low-abundance proteases.

One label-based fluorescence detection of a protease that cleaves the peptide bond between two specific amino acids

In order to detect a protease that cleaves the peptide bond between two specific amino acids via fluorescence, a synthetic peptide modified with two labels (a donor and an acceptor) for fluorescence resonance energy transfer (FRET) is commonly used. However, the preparation and optimization of a peptide for the sensitive and selective detection of a target protease are time-consuming. In this study, we report a simple method for fluorescence protease detection using a readily prepared, one label-based peptide. The fluorescence detection of botulinum neurotoxin type E light chain (BoNT/E-LC) is based on a two-step proteolytic cleavage that involves the use of BoNT/E-LC and an externally supplemented L-leucine-aminopeptidase (LAP). BoNT/E-LC cleaves the specific peptide bond between arginine and isoleucine within C-terminally 7-amino-4-methylcoumarin (AMC)-labeled oligopeptide, leaving fragmented isoleucine–AMC. Subsequently, LAP cleaves the peptide bond between isoleucine and AMC, liberating fluorescent AMC. This method does not require two label-modified peptides. Capping the oligopeptide with the D-form of tyrosine does not result in better performance in terms of detection limit, although a higher concentration of LAP can be used. The detection limit for BoNT/E-LC in both phosphate-buffered saline and commercial bottled water is 2 ng mL−1 for an incubation period of 1 h. The fluorescence detection is selective for BoNT/E-LC among the four tested BoNTs. Fluorescence detection using one label can be readily applied to any type of proteases without using FRET.