Yafeng Wu

Electrochemistry of cytochrome P450 enzyme on nanoparticle-containing membrane-coated electrode and its applications for drug sensing

In the current study, we describe an improved system to study the two-electron delivery reaction pathway of cytochrome P450, family 2, subfamily B, polypeptide 6 (CYP2B6) in vitro. In particular, a biocompatible film containing colloidal gold nanoparticles and chitosan was used to encapsulate CYP2B6 on an electrode. The electrocatalytic behaviors of CYP2B6 toward common drugs in the absence of NADHP-cytochrome P450 reductase as electron donor were studied. In an anaerobic solution, direct and reversible electron transfer between the electroactive heme center of CYP2B6 and the electrode was observed with a formal potential of -0.454 +/- 0.006 V at pH 7.4. In an air-saturated solution, an increase in the bioelectrocatalytic reduction current was observed after drug addition. The bioelectrocatalytic products were analyzed using high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS). Both results confirmed that C-hydroxylation and heteroatom release were the main pathways for CYP2B6-mediated drug oxidation, similar to what occurred in vivo. The use of immobilized proteins in nanoparticle-containing films in drug biosensing was also demonstrated.

Electrochemical Biosensing Using Amplification-by-Polymerization

A novel signal amplification strategy for electrochemical detection of DNA and proteins based on the amplification-by-polymerization concept is described. Specifically, a controlled radical polymerization reaction is triggered after the capture of target molecules on the electrode surface. Growth of long chain polymeric materials provides numerous sites for subsequent aminoferrocene coupling, which in turn significantly enhances electrochemical signal output. Activators generated electron transfer for atom transfer radical polymerization (AGET ATRP) is used in this study for its high efficiency in polymer grafting and better tolerance toward oxygen in air. 2-Hydroxyethyl methacrylate (HEMA) and glycidyl methacrylate (GMA) are examined to provide excess hydroxyl or epoxy groups for aminoferrocene coupling. A limit of detection of 15 pM and 0.07 ng/mL is demonstrated for DNA and ovalbumin, respectively. More than 7-fold signal enhancement in ovalbumin detection has been achieved comparing to the unamplified method. In addition, a more than 5 orders of magnitude of dynamic range is achieved with a linear correlation coefficient (R(2)) of 0.997 for DNA, and a more than 3 orders of magnitude with R(2) of 0.999 for ovalbumin. Together, the results show that the coupling of amplification-by-polymerization concept with electrochemical detection offers great promises in providing a sensitive and cost-effective solution for biosensing applications.

Prostate-specific antigen detection by using a reusable amperometric immunosensor based on reversible binding and leasing of HRP-anti-PSA from phenylboronic acid modified electrode.

BACKGROUND In recent years, many automated immunoassay analyzers have been developed for accurate diagnosis of various disease states and to improve effective drug administration. Amperometric immunoassay has been increasingly applied to laboratory medicine due to its ease in automation, rapid speed and low detection limits. It is important to develop reusable immunologically-sensitive elements for prostate-specific antigen (PSA) detection. METHODS The strategy for the immunosensor construction is based on the enzyme-conjugated prostate-specific antibody (HRP-anti-PSA) reversible binding with a self-assembled phenylboronic acid monolayer on gold. RESULTS After incubating an HRP-anti-PSA modified electrode in a PSA solution, a decrease in the electrocatalytic response of the HRP-anti-PSA modified electrode to the reduction of H(2)O(2) is observed. The photometric activity assays show that this decrease of the electrocatalytic response arises from the formation of immunocomplexes of HRP-conjugated anti-PSA and its antigen, not from the loss of bound HRP-anti-PSA from the electrode surface. Analytical performances and optimal conditions of the described immunosensor are also investigated. Under the optimal conditions, the amperometric immunosensor shows a linear increase of the relative intensity in 2 PSA concentration range from 2 to 15 ng/ml and 15 to 120 ng/ml, respectively. CONCLUSION This method could be used for rapid analysis of PSA and potentially other antigens.

Signal amplification cytosensor for evaluation of drug-induced cancer cell apoptosis.

Apoptosis is involved in the pathology of a variety of diseases. The measurement of apoptosis will help us to evaluate the onset of disease and the effect of therapeutic interventions. In addition, the increased demand for understanding the early stages of apoptosis is pushing the envelope for solutions in early instance real-time monitoring of death kinetics. Here we present a novel electrochemiluminescent cytosensing strategy to quantitate apoptotic cell numbers, screen some anticancer drugs, and evaluate their effects on hepatocarcinoma cell line (HepG2) cells by utilizing the human antiphosphatidyl serine antibody (APSA) conjugated Ru(bpy)(3)(2+)-encapsulated silica nanoparticle (APSA-SiO(2)@Ru) as the detection probe. HepG2 cells were easily immobilized on the arginine-glycine-aspartic acid-serine (RGDS)-multiwalled carbon nanotubes (RGDS-MWCNTs) nanocomposite by the specific combination of RGD domains with integrin receptors on the cell surface. Then APSA-SiO(2)@Ru was introduced to the surface of apoptosis cells through the specific interaction between APSA and phosphatidylserine (PS) that distributed on the outer membrane of apoptotic cells. On the basis of the signal amplification of the APSA-SiO(2)@Ru nanoprobe, the cytosensor could respond as low as 800 cells mL(-1), showing very high sensitivity. In addition, the dynamic alterations of surface PS expression on HepG2 cells in response to drugs and the cell heterogeneity were also demonstrated. The strategy presented a promising platform for highly sensitive cytosensing and convenient screening of some clinically available anticancer drugs.

Colorimetric immunosensing via protein functionalized gold nanoparticle probe combined with atom transfer radical polymerization

A novel colorimetric immunosensing strategy based on protein-modified gold nanoparticle probes combined with atom transfer radical polymerization (ATRP) technology was proposed. Gold nanoparticles (GNPs, ∼15 nm) were functionalized with antibodies through an acylamide-bond between the carboxylic group of 11-mercaptoundecanoic acid that previously self-assembled on the surface of GNPs and the amino group of the protein (here, goat anti-rabbit immunoglobulim G (anti-IgG) used as model). The surface functionalized GNPs were used for IgG capture, which introduced initiator coupled anti-IgG (Ab2*) onto the surface of GNPs through immunoreactions. Subsequently triggered polymer growth resulted in the surface graft of preformed polymer chains onto nanoparticles that altered the optical property of GNPs. A distinct color change occurred. This could be designed for IgG detection. The spectrum absorption and colorimetric detection gave a linear range of 0.5-25 ng mL(-1) with a detection limit of 0.03 ng mL(-1) for IgG. The proposed approach showed high sensitivity from both visual and absorbance measurements. In spite of the limitations of available IgG antibodies, this approach could be easily extended to the detection of other biomarkers.

Phenylboronic acid immunoaffinity reactor coupled with flow injection chemiluminescence for determination of α-fetoprotein

A reusable and sensitive immunoassay based on phenylboronic acid immunoaffinity reactor in combination with flow injection chemiluminescence (CL) for determination of glycoprotein was described. The reactor was fabricated by immobilizing 3-aminophenylboronic acid (APBA) on glass microbeads with gamma-glycidoxypropyltrimethoxysilane (GPMS) as linkage. The alpha-fetoprotein (AFP) could be easily immobilized on the APBA coated beads through sugar-boronic interaction. After an off-line incubation, the mixture of the analyte AFP with horseradish peroxidase-labeled AFP antibody (HRP-anti-AFP) was injected into the reactor. This led the trapping of free HRP-anti-AFP by the surface coated AFP on glass beads. The trapped HRP-anti-AFP was detected by chemiluminescence due to its sensitizing effect on the reaction of luminol and hydrogen peroxide. Under optimal conditions, the chemiluminescent signal was proportional to AFP concentration in the range of 10-10 0 ng m L(-1). The whole assay process including regeneration of the reactor could be completed within 31 min. The proposed system showed acceptable detection and fabrication reproducibility, and the results obtained with the present method were in acceptable agreement with those from parallel single-analyte test of practical clinical sera. The described method enabled a low-cost, time saving and was potential to detect the serum AFP level in clinical diagnosis.

Target-Triggered Polymerization for Biosensing

Because of the potential applications of biosensors in clinical diagnosis, biomedical research, environmental analysis, and food quality control, researchers are very interested in developing sensitive, selective, rapid, reliable, and low-cost versions of these devices. A classic biosensor directly transduces ligand-target binding events into a measurable physical readout. Because of the limited detection sensitivity and selectivity in earlier biosensors, researchers have developed a number of sensing/signal amplification strategies. Through the use of nanostructured or long chain polymeric materials to increase the upload of signal tags for amplification of the signal readout associated with the ligand-target binding events, researchers have achieved high sensitivity and exceptional selectivity. Very recently, target-triggered polymerization-assisted signal amplification strategies have been exploited as a new biosensing mechanism with many attractive features. This strategy couples a small initiator molecule to the DNA/protein detection probe prior to DNA hybridization or DNA/protein and protein/protein binding events. After ligand-target binding, the in-situ polymerization reaction is triggered. As a result, tens to hundreds of small monomer signal reporter molecules assemble into long chain polymers at the location where the initiator molecule was attached. The resulting polymer materials changed the optical and electrochemical properties at this location, which make the signal easily distinguishable from the background. The assay time ranged from minutes to hours and was determined by the degree of amplification needed. In this Account, we summarize a series of electrochemical and optical biosensors that employ target-triggered polymerization. We focus on the use of atom transfer radical polymerization (ATRP), as well as activator generated electron transfer for atom transfer radical polymerization (AGET ATRP) for in-situ formation of polymer materials for optically or electrochemically transducing DNA hybridization and protein-target binding. ATRP and AGET ATRP can tolerate a wide range of functional monomers. They also allow for the preparation of well-controlled polymers with narrow molecular weight distribution, which was predetermined by the concentration ratio of the consumed monomer to the introduced initiator. Because the reaction initiator can be attached to a variety of detection probes through well-established cross-linking reactions, this technique could be expanded as a universal strategy for the sensitive detection of DNA and proteins. We see enormous potential for this new sensing technology in the development of portable DNA/protein sensors for point-of-need applications.

Surface-initiated atom-transfer radical polymerization of 4-acetoxystyrene for immunosensing.

A novel immunosensing strategy based on surface-initiated atom-transfer radical polymerization (SI-ATRP) in combination with electrochemical detection is proposed. Specifically, 4-acetoxystyrene (AS) has been chosen as a monomer for ATRP due to its ability to provide acetoxyl groups, which can be converted into phenolic hydroxyl groups for electrochemical detection in the presence of tyrosinase. A controlled radical polymerization reaction of 4-acetoxystyrene at 60 °C was triggered after immobilization of initiator molecules on an electrode surface. The growth of long-chain polymeric materials increased the concentration of phenolic hydroxyl groups, which in turn significantly enhanced the electrochemical signal output. Polymerization conditions, such as temperature and duration, monomer concentration, and the catalyst/monomer ratio have been optimized. The in situ surface-initiated ATRP was confirmed by scanning electron microscope (SEM) images and X-ray photoelectron spectroscopy (XPS) analysis. Cyclic voltammetric investigation revealed a pair of well-defined oxidation and reduction peaks at 0.232 and 0.055 V, which corresponded to the redox behavior of catechol/o-quinone on the electrode surface. The proposed approach has been successfully extended to immune recognition. A detection limit of 0.3 ng mL(-1) for rabbit immunoglobulin G (IgG) as a model antigen has been achieved. Despite the limited availability of the IgG antibody, this technology might also be expanded to the detection of other proteins and DNA.

Activators generated electron transfer for atom transfer radical polymerization for immunosensing

A novel and ultrasensitive immunosensing strategy based on activators generated electron transfer for atom transfer radical polymerization (AGET ATRP) in combination with flow injection chemiluminescent (CL) and electrochemical detection was proposed. The initiator-conjugated polyclone PSA antibodies (Ab2*), prepared by coupling of N-hydroxysuccinmidyl bromoisobutyrate (initiator) with polyclone PSA antibodies (Ab2), were immobilized on the substrate surface through sandwiched immunoreactions to trigger polymerization. AGET ATRP is used for local accumulation of glycidyl methacrylate (GMA) monomers. Horseradish peroxidase (HRP) was chosen as signal species for its well-characterized chemiluminescent and electrochemical behavior, strong enzyme activity, good solubility and ease in coupling. Growth of long chain polymeric materials provided excess epoxy groups for HRP coupling, which in turn significantly increased the loading of signal molecules and enhanced the chemiluminescent and electrochemical readouts. With the proposed strategy, a detection limit of 4.0 and 1.3 pg mL(-1) was obtained for flow injection chemiluminescent and electrocatalytic measurements, respectively. A more than 13- and 14-fold enhancement in the chemiluminescent intensity and electrocatalytic current was achieved comparing to the traditional sandwiched immunoassays using HRP-conjugated antibody directly. The proposed method exhibited an efficient amplification performance for immunosensing. This paved a new way for ultrasensitive detection of cancer biomarkers.