Ultrasensitive detection of disease biomarkers using an immuno-wall device with enzymatic amplification

• Published in: Analyst (London) Vol. 144; no. 15; p. 4589
• Main Authors: Nishiyama, Keine, Kasama, Toshihiro, Nakamata, Seiya, Ishikawa, Koya, Onoshima, Daisuke, Yukawa, Hiroshi, Maeki, Masatoshi, Ishida, Akihiko, Tani, Hirofumi, Baba, Yoshinobu, Tokeshi, Manabu
• Format: Journal Article
• Language: English
• Published: England 07-08-2019
• Subjects: Acridines - chemistry; Alkaline Phosphatase - chemistry; Animals; Antibodies, Immobilized - immunology; Biomarkers - blood; C-Reactive Protein - analysis; C-Reactive Protein - immunology; Fluorescent Dyes - chemistry; Goats; Humans; Immunoassay - instrumentation; Immunoassay - methods; Lab-On-A-Chip Devices; Limit of Detection; Mice; Microfluidic Analytical Techniques - instrumentation; Microfluidic Analytical Techniques - methods; Rabbits
• ISSN: 1364-5528
• DOI: 10.1039/c9an00480g

We present an ultrasensitive immunoassay system for disease biomarkers utilizing the immuno-wall device and an enzymatic amplification reaction. The immuno-wall device consisted of 40 microchannels, each of which contained an antibody-modified wall-like structure along the longitudinal axis of the microchannel. The wall was fabricated with a water-soluble photopolymer containing streptavidin by photolithography, and biotinylated capture antibodies were immobilized on the sides through streptavidin-biotin interaction. For an assay, introducing the target biomarker and secondary and labeled antibodies produced a sandwich complex anchored on the sides of the wall. A conventional immuno-wall device uses a fluorescence-labeled antibody as a labeling antibody. To achieve an ultrasensitive detection of a trace biomarker, we used an enzyme label and amplified the signal with the enzymatic reaction with a fluorogenic substrate in the microchannel. The highest signal/background ratio was obtained by using alkaline phosphatase-labeled antibody and 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate. To evaluate the device performance, we detected human C-reactive protein (CRP) as a model biomarker. The detection limit (LOD) of CRP in phosphate-buffered saline was 2.5 pg mL
with a sample volume of 0.25 μL. This LOD was approximately 3 orders of magnitude lower than that obtained with fluorescent-dye (DyLight 650)-labeled antibody. In addition, the present device provided a wide detection range of 0.0025-10 ng mL
for CRP. We successfully developed an ultrasensitive immunoassay system with simple operation and only a small sample volume.