Enzymatic Biosensors with Integrated Thin Film a-Si:H Photodiodes
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Enzymatic Biosensors with Integrated Thin Film a-Si:H Photodiodes A. T. Pereira1,2, V. Chu1, D. M. F. Prazeres2,3 and J.P. Conde1,3 INESC Microsistemas e Nanotecnologias and IN- Institute of Nanoscience and Nanotechnology, Rua Alves Redol 9, Lisbon, Portugal 2 Centro de Engenharia Biológica e Química, IBB – Institute of Biotechnology and Bioengineering, Instituto Superior Técnico, Av. Rovisco Pais, Lisbon, Portugal 3 Dept. of Chemical and Biological Engineering, Instituto Superior Técnico, Av. Rovisco Pais, Lisbon, Portugal 1
ABSTRACT A microfabricated amorphous silicon photodiode is used to detect chemiluminescent and colorimetric horseradish peroxidase (HRP) enzymatic reactions. Detections limits of 1 nM and 1 pM of HRP are obtained for chemiluminescent and colorimetric measurements, respectively, with the reactions carried out in solution volume of 50 µL in polystyrene microwells. Surfaceadsorbed HRP can be detected with a limit of 1 fmol.cm-2 by both detection methods. Immunoassays were performed using HRP-labeled antibodies and the detection of specific antibody-antigen molecular recognition is demonstrated both in the plastic well and inside a microfluidic channel. The application of the a-Si:H/HRP system is extended by coupling HRP with oxidase enzyme systems for glucose detection and a sensitivity of 0.1 mmol/L was achieved. INTRODUCTION Horseradish peroxidase (HRP) is one of the most widely used enzymes in analytical applications and as an enzymatic label in medical diagnostics [1]. HRP can be covalently attached to biomolecules such as DNA or other proteins such as antibodies [1]. Being capable of reducing H2O2 and also some organic peroxides, HRP-based biosensors are used to monitor peroxides in several industries (e.g., pharmaceutical, dairy) [1]. Coupling HRP with H2O2producing oxidases results in a system sensitive to the oxidase substrate, enabling the monitoring of a wide range of analytes such as glucose, ethanol, cholesterol, lactate, uric acid, pyruvate, and amino acids [1]. Thin-film amorphous silicon (a-Si:H) photodiodes present several characteristics which make them suitable for biological detection applications: low dark conductivity, high internal quantum efficiency in the visible, well established microfabrication processes, and device-quality material can be achieved at low temperatures in a variety of substrates [2]. In this paper, an a-Si:H photodiode was used to detect the chemiluminescent and colorimetric products of the enzymatic reactions of a biolabel (HRP). Measurements were performed in polystyrene microtiter plate wells using 50 µL of solution and replicated in a miniaturized disposable polydimethylsiloxane (PDMS) microfluidic format which was aligned to the photodiode array chip allowing faster reactions and the use of smaller quantities of reagents. Integration of the microfluidic channel with the a-Si:H integrated photodiode permits miniaturization and multiplexing and a portable device design. Glucose detection is performed by combining glucose oxidase and HRP o
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