Fluorescence Detection of DNA Hybridization Using an Integrated Thin-Film Amorphous Silicon n-i-p Photodiode
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Fluorescence Detection of DNA Hybridization Using an Integrated Thin-Film Amorphous Silicon n-i-p Photodiode A. C. Pimentel1, R. Cabeça1,2, M. Rodrigues1,2, D.M.F. Prazeres2,3, V. Chu1, and J. P. Conde1,3 1 INESC Microsistemas e Nanotecnologias, Rua Alves Redol, 9, Lisbon, 1000-029, Portugal 2 IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, Lisbon, 1049-001, Portugal 3 Dept. of Chemical and Biological Engineering, Instituto Superior Técnico, Av. Rovisco Pais, Lisbon, 1049-001, Portugal ABSTRACT This paper presents the fluorescence detection of DNA hybridization with a surface immobilized probe using a hydrogenated amorphous silicon (a-Si:H) photosensor. This sensor integrates a SiO2 layer for DNA probe immobilization, a p-i-n a-Si:H photodiode for fluorescence detection and a fluorescence filter of hydrogenated amorphous silicon carbide (aSiC:H) to cut the excitation light. With this integrated photosensor system, a five order of magnitude difference was obtained in the signal measured at the emission wavelength and that measured at the excitation wavelength for the same incident photon flux. The fluorophore Alexa Fluor 430 was used to label the DNA target molecules and a laser at 405 nm and a photon flux of 5.7x1016 cm-2.s-1 was used as the excitation light source. The detection limit achieved for fluorophores in solution in contact with the device and for fluorophores immobilized on the device surface is 5x10-9 M and 0.4 pmol/cm2, respectively. The hybridization of the tagged DNA target with a covalently or electrostatically immobilized probe was successfully detected at a surface density of ~3 pmol/cm2. INTRODUCTION Fluorescence is one of the most commonly used methods in biology and biomedical analysis for detection of nucleic acids, proteins and cells. This method uses an external light source to excite fluorophores attached to the biomolecules of interest and measures the resultant fluorescence. Hydrogenated amorphous silicon (a-Si:H) photosensors are a promising candidate for integrated optoelectronic detection of fluorescently labeled biomolecules in microarray and labon-a-chip applications due to their high quantum efficiency in the visible light spectrum, low dark current, and low-temperature processing technology (below 250 ºC) which allows the use of substrates such as glass and polymers [1]. In previous work, fluorescence detection of immobilized DNA was demonstrated using an amorphous silicon a-Si:H sensor in a parallel contact configuration with an integrated optical filter. The detection limit of this sensor was of the order of 1x1012 molecules/cm2 [2]. Kamei et al. used an a-Si:H p-i-n photodiode with a ZnS/YF3 [3] and a SiO2/Ta2O5 [4] fluorescence filter for DNA detection in solution. The detection limits reported were 17 nM [3] and 7 nM [4]. Caputo et al [5] reported the detection of DNA using a single and a multicolor amorphous silicon p-i-n photodiode for fluorescence detection in solut
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