Biomedical subjects

Quan Cheng

Publications and source records attributed to Quan Cheng.

16 recordsLinked to original sources

PubMed2007

Recent advances in surface plasmon resonance based techniques for bioanalysis.

Surface plasmon resonance (SPR) is a powerful and versatile spectroscopic method for biomolecular interaction analysis (BIA) and has been well reviewed in previous years. This updated 2006 review of SPR, SPR spectroscopy, and SPR imaging explores cutting-edge technology with a focus on material, method, and instrument development. A number of recent SPR developments and interesting applications for bioanalysis are provided. Three focus topics are discussed in more detail to exemplify recent progress. They include surface plasmon fluorescence spectroscopy, nanoscale glassification of SPR substrates, and enzymatic amplification in SPR imaging. Through these examples it is clear to us that the development of SPR-based methods continues to grow, while the applications continue to diversify. Major trends appear to be present in the development of combined techniques, use of new materials, and development of new methodologies. Together, these works constitute a major thrust that could eventually make SPR a common tool for surface interaction analysis and biosensing. The future outlook for SPR and SPR-associated BIA studies, in our opinion, is very bright. Surface plasmon resonance (SPR) is a powerful and versatile spectroscopic method for biomolecular interaction analysis (BIA) and has been well reviewed in previous years. This updated 2006 review of SPR, SPR spectroscopy, and SPR imaging explores cutting-edge technology with a focus on material, method, and instrument development. A number of recent SPR developments and interesting applications for bioanalysis are provided. Three focus topics are discussed in more detail to exemplify recent progress. They include surface plasmon fluorescence spectroscopy, nanoscale glassification of SPR substrates, and enzymatic amplification in SPR imaging. Through these examples it is clear to us that the development of SPR-based methods continues to grow, while the applications continue to diversify. Major trends appear to be present in the development of combined techniques, use of new materials, and development of new methodologies. Together, these works constitute a major thrust that could eventually make SPR a common tool for surface interaction analysis and biosensing. The future outlook for SPR and SPR-associated BIA studies, in our opinion, is very bright.

Biological Assay↗

PubMed2006

Development of a perchlorate reductase-based biosensor for real time analysis of perchlorate in water.

Perchlorate (ClO4-) contamination of ground water is a widespread problem in the U.S., which can adversely affect human health and wildlife. Current methods for detecting and quantifying ClO4- in water are time consuming, expensive and sometimes subject to complex procedures. This study reports the construction of a ClO4- reductase-based biosensor for rapid determination of ClO4- in water. Using a 3 mm GCE (glass carbon electrode), a ClO4- sensing bio-electrode was constructed by coating an aliquot of a Dechlorosoma sp. ClO4- reductase on nafion (ion-exchange matrix) layer pre-coated on the polished surface of the GCE. The response time to ClO4- was approximately 111+/-28 s. Kinetic evaluation of the sensor response to ClO4- revealed linear increases (r2>99%) in 10 min with k values of 10.3, 24.2, 33.9 and 48.2 at 25, 50, 75 and 100 microg/L, respectively. A strong linear correlation was established between biosensor response (nA) and ion-chromatography conductivity readings (microS). Biosensor response to ClO4- was maximal at applied potential range of -0.6 to -1.0 V. ClO4- reduction was maximal in the range of 7.6 to 8.0. The ClO4- biosensor was significantly stable after repeated use (24 analyses conducted on day 1 over a 10-h period at room temperature). This study indicates great potential for the development of a portable biosensor for real time analysis of ClO4- in water.

Biosensing Techniques↗

PubMed2006

Surface plasmon resonance imaging analysis of protein-receptor binding in supported membrane arrays on gold substrates with calcinated silicate films.

A new method to fabricate supported bilayer membrane (SBM) arrays for surface plasmon resonance (SPR) imaging analysis is demonstrated in this work. Thin silicate films are produced on gold SPR substrates using layer-by-layer assembly, followed by calcination. Etching into the glassified substrates using photolithographic techniques generates nanowells of desirable size and depth. Atomic force microscopy and SPR imaging analysis show that the features are well-defined, and the etching process appears to have a surface smoothing effect. After the wells are oxidized with strong acid, vesicles spontaneously fuse onto them to form supported membranes with a high degree of lateral mobility. Fluorescence recovery after photobleaching measurements yielded a diffusion coefficient of 1.1 mum2/s. To demonstrate the feasibility for high-throughput receptor-ligand interaction analysis, binding of cholera toxin (CT) to SBM arrays containing 5 mol % ganglioside GM1 receptor was carried out with SPR imaging. The results showed excellent well-to-well reproducibility (8% RSD at 60 nM CT) and marked detection sensitivity.

Calcium↗

PubMed2006

Manipulating FRET with polymeric vesicles: development of a "mix-and-detect" type fluorescence sensor for bacterial toxin.

A simple "mix-and-detect" type of fluorescence sensor for cholera toxin (CT) is reported. The sensor consists of a BODIPY lipid dye and polydiacetylene (PDA) vesicles and utilizes the lipid insertion and FRET mechanism to offer a direct and fluorescence "turn-on" detection of the analyte. BODIPY conjugated GM1, dissolved in a Tris buffer through aggregate formation, demonstrated substantial fluorescence quenching with addition of PDA vesicle solution. The close proximity of the dye molecules to the conjugated chains as a result of lipid insertion enables energy transfer from dye to the polymer backbone, yielding the observed phenomenon. When CT is present, the binding of BO-GM1 to CT results in formation of a complex that prohibits it from membrane insertion, leading to the blocking of the quenching process. The fluorescence signal was found to be proportional to the CT concentration. The method is very simple and allows specific and sensitive detection of the protein toxin with just a few mixing steps. It can be further developed into a general sensing strategy for detection of other proteins with amplified FRET mechanism.

Biosensing Techniques↗

PubMed2006

Immunosensing of Staphylococcus enterotoxin B (SEB) in milk with PDMS microfluidic systems using reinforced supported bilayer membranes (r-SBMs).

A versatile and novel method has been developed for microfluidic immunosensing of the food-borne pathogen Staphylococcus enterotoxin B (SEB) in poly(dimethylsiloxane) (PDMS) chips. Supported bilayer membranes (SBMs) were generated by vesicle fusion in oxidized PDMS microchannels for minimizing non-specific adsorption of biomolecules. The stability of SBMs was strengthened with a streptavidin layer to make them air-stable and allow for subsequent display of the biotin-functionalized antibodies. The reinforced supported bilayer membranes (r-SBMs) are fluid, exhibiting a lateral diffusion coefficient of approximately 1.9 microm(2) s(-1), and no detectable change of mobility was found after dehydration/rehydration. This is a substantial improvement over phosphatidylcholine (PC) membranes on PDMS, which suffered a roughly 10% reduction in the mobile fraction and 30% decrease in mobility after dehydration. Non-specific protein adsorption in the membrane-treated channels was reduced 100-1000 fold as compared to PDMS surfaces without a membrane coating. A flow-based microfluidic immunosensor for SEB was developed using antibodies linked to the r-SBMs in PDMS channels, and a detection limit of 0.5 ng mL(-1) was obtained from the linear portion of the calibration curve. The microchip was applied to detection of SEB in milk, and similar response and sensitivity were obtained, demonstrating the sensor's remarkable performance for real world samples. The r-SBMs overcome the stability hurdle in SBM-modified surfaces, opening up possibilities for transport and storage of membrane-functionalized microchips in the dehydrated form without compromising the performance, and facilitating the commercialization of disposable SBM-based microdevices.

Adsorption↗

PubMed2006

Nanoscale glassification of gold substrates for surface plasmon resonance analysis of protein toxins with supported lipid membranes.

Surface plasmon resonance (SPR) spectroscopy, a powerful tool for biosensing and protein interaction analysis, is currently confined to gold substrates and the relevant surface chemistries involving dextran and functional thiols. Drawbacks of using self-assembled monolayers (SAMs) for SPR-related surface modification include limited stability, pinhole defects, bioincompatibility, and nonspecific protein adsorption. Here we report the development of stable nanometer-scale glass (silicate) layers on gold substrates for SPR analysis of protein toxins. The nanoscale silicate layers were built up with layer-by-layer deposition of poly(allylamine hydrochloride) and sodium silicate, followed by calcination at high temperature. The resulting silicate films have a thickness ranging from 2 to 15 nm and demonstrate outstanding stability in flow cell conditions. The use of these surfaces as a platform to construct supported bilayer membranes (SBMs) is demonstrated, and improved performance against protein adsorption on SBM-coated surfaces is quantified by SPR measurements. SBMs can be formed reproducibly on the silicate surface via vesicle fusion and quantitatively removed using injection of 5% Triton X-100 solution, generating a fresh surface for each test. Membrane properties such as lateral diffusion of the SBMs on the silicate films are characterized with photobleaching methods. Studies of protein binding with biotin/avidin and ganglioside/cholera toxin systems show detection limits lower than 1 microg/mL (i.e., nanomolar range), and the response reproducibility is better than 7% RSD. The method reported here allows many assay techniques developed for glass surfaces to be transferred to label-free SPR analysis without the need for adaptation of protocols and time-consuming synthetic development of thiol-based materials and opens new avenues for developing novel bioanalytical technologies for protein analysis.

Avidin↗

PubMed2006

Label-free detection methods for protein microarrays.

With the growth of the "-omics" such as functional genomics and proteomics, one of the foremost challenges in biotechnologies has become the development of novel methods to monitor biological process and acquire the information of biomolecular interactions in a systematic manner. To fully understand the roles of newly discovered genes or proteins, it is necessary to elucidate the functions of these molecules in their interaction network. Microarray technology is becoming the method of choice for such a task. Although protein microarray can provide a high throughput analytical platform for protein profiling and protein-protein interaction, most of the current reports are limited to labeled detection using fluorescence or radioisotope techniques. These limitations deflate the potential of the method and prevent the technology from being adapted in a broader range of proteomics applications. In recent years, label-free analytical approaches have gone through intensified development and have been coupled successfully with protein microarray. In many examples of label-free study, the microarray has not only offered the high throughput detection in real time, but also provided kinetics information as well as in situ identification. This article reviews the most significant label-free detection methods for microarray technology, including surface plasmon resonance imaging, atomic force microscope, electrochemical impedance spectroscopy and MS and their applications in proteomics research.

Animals↗

PubMed2005

Characterization of micropatterned lipid membranes on a gold surface by surface plasmon resonance imaging and electrochemical signaling of a pore-forming protein.

We report the fabrication and characterization of a micropatterned membrane electrode for electrochemical signaling of a bacterial pore-forming toxin, Streptolysin O (SLO) from S. pyogenes. Microcontact printing of an alkylthiol monolayer was used to fabricate an array template, onto which cholesterol-containing DMPC vesicles were fused to form lipid layer structures. The construction of the supported membranes, including pattern transfer and vesicle fusion, was characterized by in-situ surface plasmon resonance (SPR) imaging and electrochemistry. Quantitative analysis of the resulting membrane by using SPR angular shift measurements indicates that the membranes in the hydrophilic pockets have an average thickness of 8.2 +/- 0.4 nm. Together with fluorescence microscopy studies, the results suggest that this could be a mixed lipid assembly that may consist of a bilayer, vesicle fragments, and lipid junctions. The voltammetric response of the redox probe ferrocene carboxylic acid (FCA) was measured to quantify the toxin action on the supported membrane. The electrochemical measurements indicate that fusion of vesicles on the template blocked the access of FCA, whereas the injection of SLO toxin restored the redox response. The anodic peak current of FCA was found to increase with toxin concentration until a plateau was reached at 40 HU/mL. The method is highly sensitive such that 0.1 HU/mL of SLO (1.25 pM) can yield a well-defined response. In addition, it eliminates the need for a highly insulating layer in membrane sensing, which opens up new avenues in developing novel sensing interfaces for membrane-targeting proteins and peptides.

Electrochemistry↗

PubMed2005

Vesicular polydiacetylene sensor for colorimetric signaling of bacterial pore-forming toxin.

A vesicle-based polydiacetylene biosensor for colorimetric detection of bacterial pore-forming toxin streptolysin O (SLO) is reported. The sensor was constructed with three lipid constituents: glycine-terminated diacetylene lipid Gly-PCDA, cell membrane-mimicking component PC-DIYNE, and cholesterol (CHO), which serves as the bait molecule. UV irradiation led to photopolymerization of the diacetylene lipids that gave the material a blue appearance. Incubation of the vesicles with SLO from Streptococcus pyrogenes turned the vesicle solution red, and the color change was found to be correlated to SLO concentration. The optimal sensing performance was found with vesicles consisting of 71% Gly-PCDA, 25% CHO, and 4% PC-DIYNE, and a correlation relationship was obtained for 20 HU to 500 HU/mL, or 100 pM to 6.3 nM of SLO toxin. Transmission electron microscopy and dynamic light scattering was used for further characterization of the vesicular assemblies. Transmembrane pores (holes) with diameter around 30 nm were observed on the vesicle membranes, in particular on the peripheral of the membrane structures, suggesting pore formation by SLO toxin provides the driving force for the color change of the functional vesicles.

Acetylene↗

PubMed2005

Stable and fluid ethylphosphocholine membranes in a poly(dimethylsiloxane) microsensor for toxin detection in flooded waters.

Highly stable and fluid supported bilayer membranes were fabricated by fusion of positively charged ethylphosphocholine (DOPC+) vesicles into poly(dimethylsiloxane) (PDMS) microchannels for immunosensing of cholera toxin (CT) in flooded waters. Compared to phosphatidylcholine (PC) layers in the microchannels, DOPC+ membranes show exceptionally strong resistance to air-dry damage, as demonstrated by fluorescence recovery after photobleaching (FRAP) measurements and protein adsorption studies. In FRAP experiments, the mobile fraction of PC membranes was found to decrease by 10% upon drying/rehydration and the lateral diffusion coefficient decreased from 2.2 to 1.6 microm(2)/s, whereas the mobile fraction and diffusion coefficient for DOPC+ membranes remain virtually unchanged during this process. Characterization by confocal microscopy reveals that only 1% of the DOPC+ membrane in the microchannels was removed by the drying/rehydration process, as compared to 11% for PC. Protein adsorption trends indicate that the charge of DOPC+ membranes allows for tuning of solution conditions to enable the desired protein-membrane interaction to predominate at the interface. A flow-based immunoassay for bacterial toxin was developed with 5% GM1/DOPC+ membranes in PDMS channels, and a detection limit of 250 amol for CT was obtained from the calibration curves. The assay was successfully applied to detection of CT spiked in water samples from the Santa Ana River, with nearly identical response and sensitivity.

Biosensing Techniques↗

PubMed2005

Microfluidic immunoassay for bacterial toxins with supported phospholipid bilayer membranes on poly(dimethylsiloxane).

We report a heterogeneous immunoassay for cholera toxin (CT) using supported bilayer membranes (SBMs) in a poly(dimethylsiloxane) (PDMS) microfluidic device. Phosphatidylcholine membranes assembled on plasma-oxidized PDMS by vesicle fusion bring about favorable surface properties, such as improved wettability and protein resistance. Contact angle measurements show that the lipid membranes can preserve hydrophilic surfaces for hours, whereas untreated substrates rapidly undergo hydrophobic recovery. Fluorescence recovery after photobleaching performed in situ reveals that the membranes have relatively high lateral mobility. Experimental data-fitting to theoretical models yields diffusion coefficients of 1.8 +/- 0.7 microm(2)/s on PDMS and 3.4 +/- 0.8 microm(2)/s on glass. Fluorescence studies utilizing tagged proteins show that SBMs reduce nonspecific adsorption of avidin and BSA on PDMS by 2-3 orders of magnitude, as compared to that on plasma oxidized surfaces. SBMs and their protein-resistant properties are not significantly affected by long flow times, indicating good membrane stability. These studies increase our understanding of the relationship between molecular level interactions and membrane properties, allowing for development of a rapid heterogeneous immunoassay for CT in PDMS microchips with cell surface receptor molecules. Using optimized sample injection and buffer washing conditions, microfluidic immunoassay of CT is complete within 25 min, and a dynamic range over 3 orders of magnitude with a detection limit of 8 fmol of toxin is achieved.

Adsorption↗

PubMed2004

Detection of influenza virus: traditional approaches and development of biosensors.

Influenza is an acute respiratory disease caused by the influenza virus. The disease occurs annually, causing fatality in the elderly and children and billions of dollars loss in business and productivity. Traditional viral detection methods include MDCK cell culture, complement fixation, hemagglutinin-inhibition, and recently RT-PCR. Although effective, these methods generally involve labor-intensive laboratory procedures and often require trained personnel to carry them out. The development of biosensor technologies will enable rapid and specific disease diagnosis on-site so that a clinician can quickly determine whether treatment is needed. This paper reviews traditional viral assays and progress in the biosensor development for influenza virus. Recent advances in single-step direct detection using non-labeling techniques such as surface plasmon resonance, quartz-crystal microbalance, and colorimetric functional polymers are discussed.

Antiviral Agents↗

PubMed2004

Analysis of micro-contact printed protein patterns by SPR imaging with a LED light source.

We demonstrate the characterization of mu-contact printed protein patterns and analysis of protein-protein interactions by two-dimensional (2-D) surface plasmon resonance imaging (SPRi). Advancements in SPRi image quality from employing a light emitting diode (LED) as the light source are described. We show that a LED offers an ideal point source that can eliminate interference artifacts and speckles found when using a laser source. The attainable thickness resolution in fixed-angle imaging is comparable to that of a monochromatic source, providing a solid foundation for quantitative analysis with the system. The SPR imaging technique reported here affords sub-nanometer thickness sensitivity and micrometer lateral resolution, allowing for convenient studies of biomolecular interactions and surface morphologies of ultrathin films. Spatially well-defined protein patterns of bacterial toxins were obtained by microcontact printing using a polydimethylsiloxane (PDMS) stamp on a functionalized self-assembled monolayer on Au. The influence of protein concentration in the inking solution on transfer efficiency was investigated, and a nonlinear correlation was observed between the solution concentration and the amount of protein immobilized on the surface. Quantitative analysis of protein interaction was performed with toxin-specific antibody, showing a concentration-dependent relationship that verifies the retention of biological activity of the protein after printing. The study demonstrates the feasibility and effectiveness of using LEDs as light sources in SPR imaging, opening doors for developing compact SPR instruments for direct, sensitive, and label-free detection of biohazardous molecules.

Light↗

PubMed2004

Functional lipid microstructures immobilized on a gold electrode for voltammetric biosensing of cholera toxin.

Redox functionalized microstructures of diacetylene lipids containing cell surface ligand GM1 have been prepared for the construction of an electrochemical biosensor for cholera toxin from Vibrio cholerae. Incorporation of lipid molecules with disulfide functionality into the microstructures allows for firm attachment of the microstructures on a gold surface to form a sensing interface. The observed morphology of the microstructures is platelet, with size around 240 nm as determined by dynamic light scattering and transmission electron microscopy. The electrochemical response stems from electron transfer between the electrode and the redox sites on the microstructures, and the Faradaic current is influenced by the binding events of protein toxins to the ligands displayed on the crystalline surface. Electrochemical characterization indicates that electron transfer of surface ferrocene on the gold electrode is facile. Differential pulse voltammetry was used to measure the current magnitude as a function of toxin concentration, and a working range expanding from 1.0 x 10(-8) to 5.0 x 10(-7) M was obtained. Bovine serum albumin (BSA) was used as a control agent with which no interference to Faradaic response was found in the same concentration range. Atomic force microscopy (AFM) was used to characterize the morphology and distribution of microstructures on the gold surface. The effectiveness of the design for bypassing surface fouling of proteins in electrochemical detection has been demonstrated, and a binding regulated electron hopping mechanism for the observed electrochemical behavior has been proposed.

Biosensing Techniques↗

PubMed2002

Surface-bound lipid vesicles encapsulating redox species for amperometric biosensing of pore-forming bacterial toxins.

A new method is described to signal the presence of a staphylococcal toxin (streptolysin, SLO) by a membrane-based biosensor fabricated with bilayer vesicles of phosphatidylcholine lipids and cholesterol on a gold electrode. The controlled release of encapsulated redox species from the surface-bound vesicles is mediated by the pore-formation functionality of SLO, allowing amperometric detection of the targeted toxin. Effects of BSA and Triton-100 on membrane permeability and redox release have been assessed, and a thin layer electrolysis mechanism has been proposed to account for the observed voltammetric behavior. The method is applicable to the detection of functionally similar protein toxins.

Bacterial Proteins↗

PubMed2002

Morphological manipulation of bolaamphiphilic polydiacetylene assemblies by controlled lipid doping.

Morphological transformations of bolaamphiphilic polydiacetylene (L-Glu-Bis-3) lipid assemblies from helical ribbons to vesicles and flat sheets through controlled doping are described, and the role of specific lipid dopants in these processes is discussed. Upon doping with cell surface receptor G(M1) ganglioside, fluid vesicular structures start to emerge, coexisting with the micro-crystalline helical ribbons. The vesicle formation is further facilitated and stabilized by the introduction of cholesterol into the system, presumably through surface curvature variation induced by inhomogeneous distribution and dynamic clustering of G(M1) and cholesterol within the doped assemblies. Extended helical ribbons are "truncated" into patches of flat sheets when a sufficient amount of Bis-1, a structurally compatible symmetric bolaamphiphilic diacetylene lipid, is doped. The results reaffirm the important roles of packing geometry and headgroup chirality in the formation of extended helical ribbon structures. The doped assemblies of bolaamphiphiles allow for capture of intermediate structures of morphological transformation using transmission electron microscopy (TEM). A vesicle-to-ribbon transformation mechanism via lateral reorganization within relatively fluid vesicular microstructures has been suggested. Understanding of the doping-induced transformation process provides useful information for the design of advanced materials where the microscopic morphology of material is crucial to its function.

Acetylene↗