Silica nanoparticles based microfluidic immunosensor with laser-induced fluorescence detection for immunoreactive trypsin quantification

MARCO A. SEIA; PATRICIA STEGE; PEREIRA SIRLEY V.; IRMA E. DE VITO; JULIO RABA; GERMÁN A. MESSINA

ANALYTICAL BIOCHEMISTRY

ACADEMIC PRESS INC ELSEVIER SCIENCE

Lugar: Amsterdam; Año: 2014 p. 31 - 37

0003-2697

The purpose of this study is to develop a silica nanoparticle-based inmunosensor with laser-induced fluorescence (LIF) as a detection system . The proposed device was applied to quantify the immunoreactive trypsin (IRT) in Cystic Fibrosis (CF) Newborn Screening. A new ultrasonic procedure was used to extract the IRT from blood spot samples collected on filter papers. After extraction, the IRT reacted immunologically with anti-IRTmonoclonal antibodies immobilized on a microfluidic glass chip modified with 3-aminopropyl functionalized silica nanoparticles (APSN-APTES-modified glass chips). The bounded IRT was quantified by Horseradish peroxidase conjugated-anti IRT-antibody (HRP-conjugated-anti-IRT-Ab) using 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) as enzymatic mediator. The HRP catalyzed the oxidation of non fluorescent ADHP to highly fluorescent resorufin, which was measured by LIF detector, using excitation lambda at 561 nm and emission at 585 nm. The detection limits (LOD) calculated for LIF detection and for commercial ELISA test Kit were 0.87 ng mL-1 and 4.2 ng mL-1 respectively. The within- and between-assay variation coefficients for LIF detection procedure were below 6.5%. The blood spot samples collected on filter papers were analyzed with the proposed method and the results were compared with the reference ELISA method demonstrating a potential usefulness for the clinical assessment of IRT in the early neonatal period.