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ABSTRACT: An iTPA (isothermal target and signaling probe amplification) method for the quantitative detection of nucleic acids, based on a combination of novel ICA (isothermal chain amplification) and fluorescence resonance energy transfer cycling probe technology (FRET CPT), is described. In the new ICA method, which relies on the strand displacement activity of DNA polymerase and the RNA degrading activity of RNase H, two displacement events occur in the presence of four specially designed primers. This phenomenon leads to powerful amplification of target DNA. Since the amplification is initiated only after hybridization of the four primers, the ICA method leads to high specificity for the target sequence. As part of the new ICA method, iTPA is achieved by incorporating FRET CPT to generate multiple fluorescence signals from a single target molecule. Using the resulting dual target and signaling probe amplification system, even a single copy level of a target gene can be successfully detected and quantified under isothermal conditions.
Analytical Chemistry 07/2010; 82(14):5937-43. · 5.86 Impact Factor
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ABSTRACT: We describe a facile gold nanoparticle (AuNP)-mediated colorimetric method for real-time detection of target DNA in conjugation with our unique isothermal target and signaling probe amplification (iTPA) method, comprising novel ICA (isothermal chain amplification) and CPT (cycling probe technology). Under isothermal conditions, the iTPA simultaneously amplifies the target and signaling probe through two displacement events induced by a combination of four specially designed primers, the strand displacement activity of DNA polymerase, and the RNA degrading activity of RNase H. The resulting target amplicons are hybridized with gold nanoparticle cross-linking assay (GCA) probes having a DNA-RNA-DNA chimeric form followed by RNA cleavage by RNase H in the CPT step. The intact GCA probes were designed to cross-link two sets of DNA-AuNPs conjugates in the absence of target DNA, inducing aggregation (blue color) of AuNPs. On the contrary, the presence of target DNA leads to cleavage of the GCA probes in proportion to the amount of amplified target DNA and the solution remains red in color without aggregation of AuNPs. Relying on this strategy, 10(2) copies of target Chlamydia trachomatis plasmid were successfully detected in a colorimetric manner. Importantly, all the procedures employed up to the final detection of the target DNA were performed under isothermal conditions without requiring any detection instruments. Therefore, this strategy would greatly benefit convenient, real-time monitoring technology of target DNA under restricted environments.
Biosensors & bioelectronics 07/2010; 26(5):1953-8. · 5.43 Impact Factor
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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.
Biosensors & bioelectronics 09/2008; 24(5):1324-9. · 5.43 Impact Factor
