Silica nanoparticle-based microfluidic immunosensor with laser-induced fluorescence detection for the quantification of immunoreactive trypsin

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

ANALYTICAL BIOCHEMISTRY

ACADEMIC PRESS INC ELSEVIER SCIENCE

Lugar: Amsterdam; Año: 2014 vol. 463 p. 31 - 37

0003-2697

The purpose of this study was to develop a silica nanoparticle-based immunosensor with laser-inducedfluorescence (LIF) as a detection system. The proposed device was applied to quantify the immunoreactivetrypsin (IRT) in cystic fibrosis (CF) newborn screening. A new ultrasonic procedure was used toextract the IRT from blood spot samples collected on filter papers. After extraction, the IRT reacted immunologicallywith anti-IRT monoclonal antibodies immobilized on a microfluidic glass chip modified with3-aminopropyl functionalized silica nanoparticles (APSN?APTES-modified glass chips). The bounded IRTwas quantified by horseradish peroxidase (HRP)-conjugated anti-IRT antibody (anti-IRT?Ab) using 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) as enzymatic mediator. The HRP catalyzed the oxidation ofnonfluorescent ADHP to highly fluorescent resorufin, which was measured by LIF detector, using excitationlambda at 561 nm and emission at 585 nm. The detection limits (LODs) calculated for LIF detectionand for a commercial enzyme-linked immunosorbent assay (ELISA) test kit were 0.87 and 4.2 ng ml1,respectively. The within- and between-assay variation coefficients for the LIF detection procedure werebelow 6.5%. The blood spot samples collected on filter papers were analyzed with the proposed method,and the results were compared with those of the reference ELISA method, demonstrating a potential usefulnessfor the clinical assessment of IRT during the early neonatal period.