We have developed a polymer film based plasmonic device whose optical properties are tuned for measuring biological samples. The device has a circular nanohole array structure fabricated with a nanoimprint technique using a UV curable polymer, and then gold thin film is deposited by electron beam deposition. Therefore, the device is mass-producible, which is also very important for bioaffinity sensors. First the gold film thickness and hole depth were optimized to obtain the maximum dip shift for the reflection spectra. The dip shift is equivalent to the sensitivity to refractive index changes at the plasmonic device surface. We also calculated the variation in reflection spectra by changing the above conditions using the finite-difference time domain method, and we obtained agreement between the theoretical and experimental curves. The nanohole periodicity was adjusted from 400 to 900 nm to make it possible to perform measurements in the visible wavelength region to measure the aqueous samples with less optical absorption. The tuned bottom filled gold nanohole array was incorporated in a microfluidic device covered with a PDMS based microchannel that was 2 mm wide and 20 μm deep. As a proof of concept, the device was used to detect TNF-α by employing a direct immunochemical reaction on the plasmonic array, and a detection limit of 21 ng mL(-1) was obtained by amplification with colloidal gold labeling instead of enzymatic amplification.
This paper reports the development and characterization of a highly sensitive enzyme linked immunosorbent assay realized by the electrogenerated chemiluminescence (ECL) detection of a thiol monolayer formed by an enzyme labeled antibody. We used two monoclonal anti tumor necrosis factor-alpha (TNF-alpha) antibodies for a sandwich immunoassay. One was a capture antibody, and the other was a detection antibody labeled with an enzyme via an avidin-biotin interaction. Acetylcholinesterase was used as the labeling enzyme to convert acetylthiocholine to thiocholine. Then the thiocholine was collected on a gold electrode surface by gold-thiol binding. A bright and distinctive emission was observed at 1150 mV (vs Ag-AgCl) on the gold electrode with a thiocholine monolayer as a coreactant in the presence of tris(2,2'-bipyridyl)ruthenium complex. This method can greatly enhance the immunoassay signal since a large number of coreactant molecules can be generated by the enzymatic reaction, which is advantageous compared with a previously reported ECL based immunoassay that directly labels the detection antibody with a coreactant or luminophore. In addition, a surface accumulated coreactant is superior to the previously reported coreactant system in a bulk solution, because ECL emission occurs only very close to an electrode surface. As a result, high sensitivity and a low detection limit of 0.2 pM (3.4 pg/mL) TNF-alpha were achieved with excellent reproducibility by optimizing the conditions for the immuno-reaction, thiocholine accumulation, and ECL generation.
Cytosine methylation in DNA was determined by an enzyme linked immunosorbent assay (ELISA) with electrochemiluminescence (ECL) detection and employed for the DNA methylation assay of a long and real genomic sample for the first time. The developed method employed an antimethyl cytosine antibody labeled with acetylcholinesterase, which was added to recognize single methylated cytosine in a DNA oligomer. The acetylcholinesterase converted acetylthiocholine (substrate) to thiocholine (product), which was accumulated on a gold electrode surface via gold-thiol binding. This surface accumulated preconcentration made it possible to observe bright and distinctive ECL by applying a potential to the gold electrode in the presence of a tris(2,2-bipyridyl)ruthenium complex luminophore when the analyte DNA contained a methylation region. Methyl-cytosine was measured quantitatively in the 1-100 pmol range, which exhibits sufficiently high sensitivity to achieve real DNA measurements without amplification by a polymerase chain reaction (PCR). The proposed ECL method also exhibited high selectivity for methyl-cytosine against nonmethylated cytosine, guanine, thymine, and adenine nucleotides. Finally, original and methylated DNA samples were clearly distinguished with our method using a real DNA bacteriophage sample (48,502 base pairs).
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