We demonstrate a highly sensitive detection of AFP (α-fetoprotein) protein (liver cancer marker) in human serum using the LSPR biosensor. Gold metal nanodot array (MNA) on a glass wafer were fabricated by UV nanoimprint lithography (NIL). After the NIL process using a film stamp and the removal of residual layer via oxygen plasma etching, metal films were deposited using an electron-beam evaporator, followed by the lift-off step. Consequently, the gold MNA was realized on 5-inch glass wafer and the pitch, diameter and height of MNA were 300nm, 150 nm and 20 nm, respectively. We employed observation of LSPR spectra via back-reflection, which provides a stable measurement of LSPR because a probe light does not pass a bio-sample. In addition, one channel among two flow channels was used a control channel, the MNA surface in which was modified with bovine serum albumin, not antibody. After antigen-antibody reaction, the enzyme/precipitation was employed on the MNA (Nano-ELISA). As a result, we could detect AFP in 50L human serum with limit of detection (LOD) of 0.7 zeptomole (10<sup>-21</sup> mole).
In this study, gold nanoplasmonic biosensors using localized surface plasmon resonance (LSPR) were fabricated for the diagnosis of cancer. We optimized the structures of the gold nanodot array (GNA) via the experiments for the optical characteristics. In addition, the nanoimprint lithography was employed for realizing nanoplasmonic structures, which is a more efficient technique for mass production than nanolithography such as electron beam lithography (EBL) or focused ion beam (FIB) lithography that is a quite intricate, time-consuming and expensive process.
After the UV nanoimprinting process using a film stamp and metal films were deposited using an electron-beam evaporator, followed by the lift-off step. Consequently, the nanoplasmonic MNA was realized on 5-inch glass wafer and the pitch, diameter and height of MNA were 300nm, 150 nm and 20 nm, respectively. The wavelength of nanoplasmonic resonance peak represented from the MNA sensors was about 740nm under aqueous ambient.
The capture antibodies of the lung and the pancreas cancer marker, respectively, were immobilized on the surfaces of MNA sensor. Using a compact fiber-optic spectrometer and a reflection optical probe, we were able to confirm the binding of cancer markers with their antibodies due to the immunoreactions between each cancer marker and its corresponding antibody on the sensor surfaces. The amount of the cancer markers in serum were analyzed through the observation of nanoplasmonic resonance wavelength-shift on the reflection spectra. To amplify a sensitivity of detection demonstrated by the nanoplasmonic resonance peak shift, we applied enzyme-precipitation reaction on the surface of MNA biosensor. The enzyme-catalyzed precipitation method in the GNA biosensor could be extended to detect other clinical biomarkers at extremely low concentrations in actual clinical samples.
Herein, we have developed a label-free and homogeneous fluorescence resonance energy transfer (FRET) immunoassay
for the detection of neopterin (NPT), which is an early and valuable biochemical marker of cellular immunity. Owing to
intrinsic fluorescence properties of antibody and NPT, anti-NPT antibody (anti-NPT) and analyte played roles as the
respective donor and acceptor in the FRET immunoassay. As the concentration of NPT increases, the fluorescence
intensity at ~350 nm decreases owing to the formation of increasing amounts of the anti-NPT/NPT complex in which
FRET takes place. The assay system was found to display a high specificity and a low detection limit (0.14 ng mL<sup>-1</sup>) for
NPT. A practical application of the FRET immunoassay system was demonstrated by its use in the detection of NPT in
spiked human serum samples. The observations made in these efforts show that the homogeneous FRET immunoassay
strategy, which requires a simple sample preparation procedure, serves as a powerful tool for the rapid and sensitive
quantitative determination of NPT.
Multichannel images of 11-Mercaptoundecanoic acid and 11-Mercapto-1-undecanol self-assembled monolayers (SAMs) together with a biospecific interferon-gamma (IFN-gamma)/anti-IFN-gamma antibody immunoreaction were observed by two-dimensional surface plasmon resonance (2D-SPR) imaging system. Patterning process for SAM was simplified by exploiting direct photooxidation of thiol bonding (photolysis) instead of conventional photolithography. Sharper images were resolved by using a white light source in combination with a narrow bandpass filter, minimizing the diffraction patterns on the images. The line profile calibration of the image contrast caused by different resonance conditions at each points on the sensor surface enabled us to discriminate the monolayer thickness in a sub-nanometer scale. For protein patterning, a precipitation scheme induced by biocatalytic reaction was implied for the signal amplification. Specific binding of IFN-gamma antigen with surface-immobilized antibody was found detectable down to the concentration of 1 ng/mL.