Kohei Nakamoto

Determination of DNA Methylation Using Electrochemiluminescence with Surface Accumulable Coreactant

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).

Development of a mass-producible on-chip plasmonic nanohole array biosensor

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.

Electrochemical Surface Plasmon Resonance Measurement Based on Gold Nanohole Array Fabricated by Nanoimprinting Technique

In this paper, we describe our development of an electrochemical surface plasmon resonance (EC-SPR) measurement device based on a bottom-filled gold nanohole array. The polymer based gold nanohole array was fabricated with a UV nanoimprint technique and electron beam gold deposition. Direct reflection mode measurement was used to monitor the SPR dip in the reflection spectra. A cyclic voltammogram was also operated by using the standard three electrodes containing working electrode having a gold nanohole array and counter and reference electrodes. The gold nanohole array was modified with an osmium-poly(vinylpyridine)-wired horseradish peroxidase (Os-gel-HRP) film, and its redox state induced by the change in potential was monitored simultaneously. The redox state of the local film was obtained simply by scanning the sample substrate stage. The substrate modified with Os-gel-HRP film was incorporated in a microfluidic chip, and then the hydrogen peroxide was determined in terms of the redox change in the Os complex mediator from the slope of the SPR dip shift. The linear relation of hydrogen peroxide from 10 to 250 μM was successfully monitored, and a high conversion efficiency was realized.

One-chip biosensor for simultaneous disease marker/calibration substance measurement in human urine by electrochemical surface plasmon resonance method

We have developed a miniaturized electrochemical surface plasmon resonance biosensor for measuring two biomolecules that have very different molecular sizes, one is transferrin (MW=75 kDa) as a disease marker protein, the other is creatinine (MW=113) as a calibration marker for the accurate measurement of human urinary samples. The sensor has a PDMS based microchannel that is 2 mm wide and 20 μm deep. Two gold films were integrated in the microchannel; one was modified with anti-transferrin antibody for immuno-reaction, and the other was modified with osmium-poly-vinylpyridine wired horseradish peroxidase (Os-gel-HRP). We further immobilized a tri-enzyme layer of creatininase, creatinase and sarcosine oxidase in order to measure creatinine by converting it to hydrogen peroxide in the upstream channel. We measured the transferrin concentration from the refractive index change involved in an immuno-complex formation, and we were simultaneously able to measure creatinine by employing the refractive index change in the Os-gel-HRP caused by oxidation with the hydrogen peroxide produced from creatinine by the tri-enzyme. The effects of ascorbic acid and uric acid in urine samples were sufficiently eliminated by adding ascorbate oxidase and uricase to the urine samples during sampling. We were able to measure two analyte concentrations within 15 min by one simple injection of 50 μL of diluted human urine into our sensor. The detectable transferrin and creatinine ranges were 20 ng/mL to 10 μg/mL, and 10 μM to 10 mM, respectively, which are sufficient levels for clinical tests. Finally, we compared the results obtained using our sensor with those obtained with a conventional immunoassay and the Jaffe method. We obtained a similar trend that can reduce the fluctuation in the urinary transferrin concentration from three different samples by calibrating the creatinine concentration.