Au nanoparticles fabricated on glass substrate with coated Cr layer in 5 nm thickness under the Au as an adhesive layer. Two fabrication techniques were employed. One is chemical self-assembled monolayer (also called sphere lithography), and the other is focused ion beam direct milling. The former has advantages of low cost and simple operation facilities, but shortages of worse uniformity in both size and dimension. The latter has no limitation of shape, and thus more freedom for the particles design. An experimental biosensing system was designed and set up that can realize localized surface plasmon resonance (LSPR)-based immunoassay in-vivo. In other words, the detection can be carried out for the molecule such as antibody and antigen still involved/mixed in their original chemical solution instead of the conventional dried molecule on the biochips for the LSPR-based biosensing. Therefore, the detected results are reliable because the detection environment is the same as the actual case. Measurement error caused by variation of the biological environment is avoided accordingly. It can realize localized detection over a specific area as small as micron scale. Even single cell/molecular detection is possible in the case of the bio-samples with low concentration and detected using the objective lens with large NA.

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