Trace Biomolecule detection system
Date Issued
2010
Date
2010
Author(s)
Lin, I-Ting
Abstract
Biomolecule detection system has been rapidly developed, and it has also been generally applied onto early diagnosis of cancer. Commonly seen system such as immunoradiometric assay(IRMA)、fluorescence immunoassay(FIA)、chemiluminescence immunoassay(CLIA)、enzyme-linked immunoassay(ELISA)、immuno-polymerase chain reaction(Immuno-PCR) are all functioning effectively. Among them, there is a system which is called immuno-PCR is working through specific binding between antibody in the system and the antigen/biomplecules. The antigen which is captured will be combined with detection antibody –DNA fragment complex in the system, and the DNA fragment can be duplicated by PCR technology to enlarge the signal. Therefore, the sensitivity of immuno-PCR is much better than the commonly used ELISA system.
So far we have known that immuno-PCR is to fix antibody on glass substrate by any means in order to catch antigen. However, the hydrophilic antigen or proteins are easily attached to hydrophilic glass substrate, and cause non-specific binding which leads to the inaccuracy in examination result.
To improve the accuracy in examination result, a layer of hydrophobic diamond film has been grown on silicon substrate by MPCVD to decrease non-specific binding, subsequently the multi-walled carbon nanotubes are grown on it by HFCVD. After that the substrate processed an acid treatment, and then connected with PEG bis-amine, which can decrease non-specific binding by its steric repulsion, and the repulsion due to its negative electricity, which caused a waving effect . Moreover, it was connected to the C-terminal of α-1 antitrypsin antibody, and let the N-terminal of antibody can still remain active and caught the corresponded α-1 antitrypsin protein. After that we added in the detection antibody, that is, biotinylated α-1 antitrypsin antibody, which could be captured by the α-1 antitrypsin protein on the substrate. At the same time, the detection antibody was connected to the biotinylated DNA fragment through streptavidin, therefore we could amplify the signal by PCR.
In order to know the sensitivity and specificity of the system, α-1 antitrypsin proteins with different concentration have been detected. Polylysine has also been added in to compare that if the non-specific binding has diminished or not.
The experiment result turns out that the diamond film which is under 300 sccm hydrogen, 20 sccm methane, 1.5 sccm nitrogen, 100 torr chamber pressure, 1200W microwave power, 180 minutes growing time and processed for 1 hour heat treatment under 580°C demonstrates best quality of hydrophobicity and is also better for following growth of carbon nanotubes, while under the condition of 140 sccm nitrogen, 17.5 sccm C2H2, growing temperature 800°C, precursor sublimed temperature 200°C, growing time 120 minutes can grow the carbon nanotubes which is longer, has larger specific surface area, with uniform thickness, and the directionality. Also, from the experiment result, it shows that the detection limit of the trace biomolecule detection system built up by this research is about 0.02 μg/ml, and the unwanted signal will not appear if the concentration of non-specific protein is less than 100 μg/ml.
Subjects
diamond film
carbon nanotubes
antibody-antigen specific binding
PEG bis-amine
PCR
SDGs
Type
thesis
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