Gu Yoo

A carbon nanotube metal semiconductor field effect transistor-based biosensor for detection of amyloid-beta in human serum.

We have developed a carbon nanotube (CNT) film-based biosensor with a metal semiconductor field effect transistor structure (MESFET). A gold top gate was deposited on the middle of the CNT channel and probe antibodies were immobilized on the gold top gate with an antibody-binding protein, protein G or Escherichia coli outer membrane (OM) with autodisplayed Z-domains of protein A. These CNT-MESFET biosensors exhibited a higher sensitivity than the CNT-FET biosensor with probe antibodies immobilized using a chemical linker, since the orientation of immobilized antibodies was controlled by the antibody-binding proteins. In addition, nonspecific binding was effectively inhibited by E. coli OM. Using the CNT-MESFET biosensors with E. coli OM containing Z domain, we detected amyloid-β (Aβ) in human serum, one of the biomarkers for early diagnosis of Alzheimers disease. Aβ at the level of 1 pg/mL in human serum could be measured in real-time and without labeling, which was lower than a limit of detection for plasma Aβ using an enzyme-linked immune sorbent assay. These results suggested that our CNT-MESFET biosensors might be applicable for an early diagnosis of Alzheimers disease.

Immobilization of E. coli with autodisplayed Z-domains to a surface-modified microplate for immunoassay

Escherichia coli with autodisplayed Z-domains was reported to improve the sensitivity of immunoassays by the orientation control of antibodies. In this work, a sensitive microplate-based immunoassay is presented by immobilizing E. coli cells to a surface-modified microplate. The microplate was prepared by coating parylene-H film with formyl groups, and then covalently coupling poly-L-lysine to the parylene-H film. The E. coli cells were bound to the microplate by charge interactions between the negatively charged E. coli outer membrane and the positively charged microplate surface. In this work, the preparation of the microplate coated with poly-L-lysine is presented. The immobilization efficiency of E. coli to the modified surface was estimated to be far higher than non-specific interaction by fluorescence microscope and the optical transmittance of the modified microplate was measured to be feasible for immunoassay. The microplate-based immunoassay is demonstrated to be feasible for medical diagnosis of inflammatory diseases by using C-reactive protein as a target analyte for the medical diagnosis of inflammatory diseases.

Microarray based on autodisplayed Ro proteins for medical diagnosis of systemic lupus erythematosus (SLE).

A microarray-based immunoassay for the detection of autoantibodies against Ro protein was developed using Escherichia coli with autodisplayed Ro proteins (Ro(+)-E. coli). Patient serum usually contains various antibodies against the outer membrane components of E. coli as well as autoantibodies against the Ro protein. Therefore, the conventional immunoassay based on Ro(+)-E. coli requires both wild type E. coli (blank test) and Ro(+)-E. coli, and both strains of E. coli must be prepared in situ for each individual test serum. In this study, we tested the feasibility of using several types of animal sera as a replacement for individual human sera. An immunoassay without the blank test was developed using Ro(+)-E. coli by (1) blocking with rabbit serum, and (2) cleaving the Fc region from antibodies using papain. Modified E. coli with autodisplayed Ro protein was immobilized to a surface-modified microplate and the applicability of the immunoassay without the blank test was demonstrated using sera from patients with systemic lupus erythematosus (SLE). Using this approach, a microarray-based fluorescence immunoassay with immobilized Ro(+)-E. coli was able to detect anti-Ro autoantibodies in SLE patient sera with high specificity and selectivity and improved efficiency.

SPR biosensor based on immobilized E.coli cells with autodisplayed Z-domains

The Z-domains of protein A was expressed as a fusion protein at the outer membrane of E.coli by using the autodisplay technology. Because of the specific affinity towards the Fc region of immunoglobulins (IgG’s), the Z-domains have been used for the orientation control of antibodies in order to improve the sensitivity of immunoassays. In this work, the E.coli with autodispalyed Z-domains was immobilized to the SPR biosensor by the charge interaction. The surface modification was carried out by covalent layering of the poly-L-lysine with amino groups to the parylene-H film with formyl groups. And then, the negatively charged E.coli cells were immobilized by charge interaction with the positivley charged of poly-L-lysine. The effectiveness of this layer for the immobilization of E.coli was estimated by counting the number of E.coli cells in comparison with the bare gold surface and the poly-L-lysine coated gold surface. For the test of feasibility of the immobilized E.coli cells to SPR biosensor, the stability of immobilized E.coli cells was estimated by treatment of salt solutions at the known concentrations to the immobilized E.coli cells which were bound through the charge interaction. From this test, the E.coli cells immobilized to the parylene-H film with poly-L-lysine coating were determined to be stable at the salt concentration of human serum. Then, the applicability of the immobilized E.coli cells with autodisplayed Z-domains was demonstrated by detection of C-reactive protein (CRP). The effect of orientation control with autodisplayed Z-domains was estimated by comparing the sensivities by immobilization through the physical adsorption and charge interaction to poly-L-lysine coated layer.

Flow cytometric immunoassay using E. coli with autodisplayed Z-domains

Recently, we reported a highly sensitive immunoassay using Escherichia coli cells with autodisplayed Z-domains. In this work, E. coli cells with autodisplayed Z-domains were applied to the flow-cytometry-based simultaneous detection of multiple analytes. The E. coli cells were doubly transfected to express a fluorescent protein (tdTomato) in the cytosol and the autodisplayed Z-domains on the outer membrane. By using E. coli cells with only the autodisplayed Z-domains, immunoassay of multiple analytes could be performed simultaneously on the same sample. Flow cytometry can be used to identify the immunoassay type by simultaneously detecting the fluorescence signal from the cytosol (tdTomato) and the fluorescence from the outer membrane, enabling the quantification of bound analytes after treatment with additional fluorescently labeled antibodies. To demonstrate the immunoassay of multiple analytes by using flow cytometry, human hepatitis B virus surface antigen (HBsAg) and C-reactive protein (CRP), a broad spectrum inflammation marker, were used as model analytes.

Magnetic-bead-based immunoassay using E. coli cells with autodisplayed Z-domains.

Escherichia coli cells with autodisplayed Z-domains could increase the sensitivity of immunoassays by immobilizing antibodies in a controlled orientation. In the work presented here, E. coli cells with autodisplayed Z-domains were immobilized to magnetic beads for subsequent immunoassay. In comparing conventional immunoassay using the E. coli cells with autodisplayed Z-domains, the magnetic-bead-based immunoassay improved immunoassay efficiency by minimizing the loss of E. coli cells during repeated centrifugation steps during washing. For the immobilization of E. coli cells to magnetic beads, the magnetic beads were modified with poly-l-lysine to bind to negatively charged E. coli cells. During the surface modification process, physical parameters such as the surface charge and size of the magnetic beads were analyzed to confirm the formation of E. coli-magnetic bead complexes. To test the feasibility of the magnetic-bead-based immunoassay, horseradish peroxidase (HRP) was used as a model analyte, and a biomarker for inflammatory diseases, C-reactive protein (CRP), was used for a demonstration of an application in medical diagnosis.

Ultrasonic isolation of the outer membrane of Escherichia coli with autodisplayed Z-domains.

The outer membrane of Escherichia coli was previously isolated as a liposome-like outer membrane particle using an enzymatic treatment for lysozymes; for immunoassays, the particles were subsequently layered on solid supports via hydrophobic interactions. This work presents an enzyme-free isolation method for the E. coli outer membrane with autodisplayed Z-domains using ultrasonication. First, the properties of the outer membrane particle, such as the particle size, zeta potential, and total protein, were compared with the properties of particles obtained using the previous preparation methods. Compared with the conventional isolation method using an enzyme treatment, the ultrasonic method exhibited a higher efficiency at isolating the outer membrane and less contamination by cytosolic proteins. The isolated outer membrane particles were layered on a gold surface, and the roughness and thickness of the layered outer membrane layers were subsequently analyzed using AFM analysis. Finally, the antibody-binding activity of two outer membrane layers with autodisplayed Z-domains created from particles that were isolated using the enzymatic and ultrasonic isolation methods was measured using fluorescein-labeled antibody as a model analyte, and the activity of the outer membrane layer that was isolated from the ultrasonic method was estimated to be more than 20% higher than that from the conventional enzymatic method.

Electrochemical Analysis of Autodisplayed Adrenodoxin (ADX) on the Outer Membrane of E. coli

In this work, adrenodoxin (Adx) was expressed on the outer membrane of E. coli by autodisplay and then the iron-sulfur cluster was incorporated into apo-Adx by an anaerobic reconstitution process. For the determination of the redox potentials of the iron-sulfur clusters of the autodisplayed Adx, E. coli cells with autodisplayed Adx were immobilized on a gold electrode modified with a self-assembled monolayer of mercaptoundecanoic acid (MUA). From the repeated cyclic voltammetry (CV) analysis, the E. coli (10mM HEPES buffer, pH7.0) with autodisplayed Adx showed significant changes in shape with an oxidation peak at +0.4V (vs. Ag/AgCl) and a reduction peak at -0.3V (vs. Ag/AgCl) after the reconstitution process for the incorporation of the iron-sulfur cluster. From the repeated CV analysis in the reduction and oxidation potential ranges, the iron-sulfur clusters of the autodisplayed Adx were observed to undergo reversible redox reactions via direct electron transfer to the MUA-modified gold electrode.

FACS-based immunoassay of troponin-I using E. coli cells with autodisplayed Z-domains

Fluorescence-activated cell sorter (FACS)-based immunoassays using E. coli cells with autodisplayed Z-domains were performed to (1) improve the sensitivity of the immunoassay through orientation control of antibodies with autodisplayed Z-domains and (2) minimize the required amount of analyte by using FACS to measure the fluorescent signal from individual E. coli cells. The expression (autodisplay) of Z-domains on the outer membrane of E. coli was confirmed by fluorescence image analysis, and the homogeneous distribution of autodisplayed Z-domains was presented by SEM image analysis after treatment with antibodies labeled with gold nanoparticles (diameter of 15 nm). As FACS measures the fluorescent signal from individual E. coli cells, the optimal FACS parameters for the effective detection of fluorescently labeled E. coli cells were determined, and the minimum amount of analyte required for the E. coli cell-based immunoassay was identified using two model immunoassay configurations. Finally, the medical diagnosis of heart infarction with a biomarker called troponin-I using the E. coli cell-based immunoassay with FACS analysis was demonstrated.

Optimization of a FACS based-immunoassay using E. coli autodisplaying Z-domains

We developed a fluorescence-activated cell sorter (FACS)-based immunoassay using Escherichia coli with autodisplayed Z-domains as a solid support for detection antibodies as well as a method to optimize the signal-to-noise ratio of fluorescence signals from E. coli. To improve the sensitivity of FACS measurements by eliminating artifactual signals from unrelated small particles in the sample mixture, E. coli with autodisplayed Z-domains was modified to express enhanced green fluorescence protein (eGFP) in the cytosol. By selectively gating the measurement channel for eGFP in the cytosol, we were able to discriminate between green fluorescent E. coli and artifact particles. We confirmed the efficacy of this optimization method for FACS-based immunoassay using several model reactions. Finally, as proof of concept, we used the FACS-based immunoassay and our optimization method for the medical diagnosis of human hepatitis B virus surface antigen.