Escherichia coli with autodisplayed Z-domain of protein A for signal amplification of SPR biosensor
ABSTRACT Generally, an immunoaffinity SPR biosensor detects a target analyte in a sample through highly selective adsorption by using the antigen-antibody interaction. For improving the sensitivity, various kinds of particles have been added to the already bound analytes on the SPR biosensor (sandwich assay). In this work, signal amplification was demonstrated by the expression of the IgG-binding Z-domain of protein A on the outer membrane of Escherichia coli via "Autodisplay". The amount of Z-domain of protein A expressed on the outer membrane was calculated to be 280,000 molecules per cell. In addition, the IgG-binding ability of the expressed protein was characterized using FACS analysis. The signal amplification of the SPR biosensor was performed in the sandwich assay format using a model of horseradish peroxidase (HRP); the limit of detection was determined to be significantly improved from 1 microg/ml to 1 ng/ml. Finally, myoglobin analysis was demonstrated for the medical diagnosis of cardiac diseases. The detection limit was estimated to be improved from 10 ng/ml to <1 ng/ml. These results show that Z-domain-displaying E. coli can be successfully used for the signal amplification of immunoaffinity biosensors, thereby improving the sensitivity and the limit of detection.
- SourceAvailable from: Joachim Jose
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- "One of the most convenient features of autodisplay is the membrane anchoring domain, the ␤-barrel, which is not covalently linked to the cell envelope as it is in other display systems, but instead is motile within the plane of the outer membrane. If the displayed passenger domains have affinity to each other, this will lead to a passenger driven or self-driven dimerization or multimerization as it has been shown in addition for SDH (Jose and von Schwichow, 2004a) (Fig. 4), nitrilase (Detzel et al., 2011), prenyltransferase (Kranen et al., 2011), or the lacZ domain of protein A (Jose et al., 2009). By electron spin resonance experiments it was shown that when it reached the surface, the Adx dimer was devoid of the iron-sulfur group and hence inactive (Jose et al., 2001). "
ABSTRACT: To display an enzyme on the surface of a living cell is an important step forward towards a broader use of biocatalysts. Enzymes immobilized on surfaces appeared to be more stable compared to free molecules. It is possible by standard techniques to let the bacterial cell (e.g. Escherichia coli) decorate its surface with the enzyme and produce it on high amounts with a minimum of costs and equipment. Moreover, these cells can be recovered and reused in several subsequent process cycles. Among other systems, autodisplay has some extra features that could overcome limitations in the industrial applications of enzymes. One major advantage of autodisplay is the motility of the anchoring domain. Enzyme subunits exposed at the cell surface having affinity to each other will spontaneously form dimers or multimers. Using autodisplay enzymes with prosthetic groups can be displayed, expanding the application of surface display to the industrial important P450 enzymes. Finally, up to 10⁵-10⁶ enzyme molecules can be displayed on a single cell. In the present review, we summarize recent achievements in the autodisplay of enzymes with particular attention to industrial needs and process development. Applications that will provide sustainable solutions towards a bio-based industry are discussed.Journal of Biotechnology 04/2012; 161(2):92-103. DOI:10.1016/j.jbiotec.2012.04.001 · 2.87 Impact Factor
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- "Recently, we reported autodisplayed Z-domains on E.coli can highly improve the sensitivity of immunoassays through the orientation control of detection antibodies   . As shown in Fig. 1, the immunoassay can be performed on E.coli cells with autodisplayed Z-domains and the assay result can be estimated by using optical density (OD) measurement at a specific wavelength . "
ABSTRACT: Recently, we reported autodisplayed Z-domains on Escherichia coli can highly improve the sensitivity of immunoassays through the orientation control of detection antibodies. In this work, the amperometric analysis was applied to the immunoassay based on E. coli cells with autodisplayed Z-domains. For amperometric analysis, the reduction current of 3,5,3′,5′-tetramethylbenzidine (TMB) was measured at the potential of −50 mV and the correlation between the reduction current and the OD value was estimated to be 28 nA/OD and the limit of detection was calculated to be 0.07 in OD unit. In this work, the feasibility of the immunoassay based on amperometric analysis was presented by the quantification of HRP as a model analyte. For the demonstration of medical diagnosis, C-reactive protein (CRP) was detected by using electrochemical ELISA based on E. coli with autodisplayed Z-domains.Procedia Engineering 12/2011; 175:46–52. DOI:10.1016/j.snb.2011.11.064
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ABSTRACT: 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.BioChip journal 09/2012; 6(3). DOI:10.1007/s13206-012-6304-3 · 1.09 Impact Factor