A detailed characterization of the performances of amorphous silicon photodiodes in the detection of chemiluminescent signal is carried out. Comparison with commercial CCD acquisition system has been done as benchmark. The underlying idea is the development of stand-alone and compact micro-total-analysys-systems (μ-TAS) that do not need bulky and expensive equipment for their operation as external focusing optics and excitation sources. The photosensor is p-i-n structures deposited by Plasma Enhanced Chemical Vapour Deposition on a glass substrate covered with a transparent conductive oxide that acts as bottom electrode and window layer for the light impinging through the glass. A PDMS layer with wells has been fabricated using an aluminum mold and bonded on the glass substrate with a well aligned with a photosensor. The experiments have been performed by filling a well with solutions containing different quantities of horseradish peroxidase. A good linearity of the photosensor response is observed across the entire measurement range that spans over three orders of magnitude. The system detection limit is 70 fg/μL. A very good agreement between results achieved with conventional off-chip CCD detection and the on-chip photodiode has been observed. Experiments with target molecules immobilized on a functionalized glass surface have been also performed in microfluidic regime, confirming the validity of the proposed integrated approach based on a-Si:H technology.
KEYWORDS: Receptors, Proteins, Resonance energy transfer, Bioluminescence, Luminescence, Energy transfer, Green fluorescent protein, In vitro testing, Molecular mechanisms, Molecules
Estrogen receptor (ER) is a ligand-activated transcriptional factor, able to dimerize after activation and to bind specific DNA sequences (estrogen response elements), thus activating gene target transcription. Since ER homo- and hetero-dimerization (giving a-a and a-b isoforms) is a fundamental step for receptor activation, we developed an assay for detecting compounds that induce human ERa homo-dimerization based on bioluminescence resonance energy transfer (BRET). BRET is a non-radiative energy transfer, occurring between a luminescent donor and a fluorescent acceptor, that strictly depends on the closeness between the two proteins and can therefore be used for studying protein-protein interactions.
We cloned ERa coding sequence in frame with either a variant of the green fluorescent protein (enhanced yellow fluorescent protein, EYFP) or Renilla luciferase (RLuc). Upon ERa homo-dimerization, BRET process takes place in the presence of the RLuc substrate coelenterazine resulting in EYFP emission at its characteristic wavelength. The ER alpha-Rluc and ER alpha-EYFP fusion proteins were cloned, then the occurrence of BRET in the presence of ER alpha activators was assayed both in vivo, within cells, and in vitro, with purified fusion proteins.
The use of bio- and chemiluminescence for the development of quantitative binding assays offers undoubted advantages over other detection systems, such as spectrophotometry, fluorescence, or radioactivity. Indeed, bio- and chemiluminescence detection provides similar, or even better, sensitivity and detectability than radioisotopes, while avoiding the problems of health hazards, waste disposal, and instability associated with the use of radioisotopes. Among bioluminescent labels, the calcium-activated photoprotein aequorin, originally isolated from Aequorea victoria and today available as a recombinant product, is characterized by very high detectability, down to attomole levels. It has been used as a bioluminescent label for developing a variety of highly sensitive immunoassays, using various analyte-aequorin conjugation strategies. When the analyte is a protein or a peptide, genetic engineering techniques can be used to produce protein fusions where the analyte is in-frame fused with aequorin, thus producing homogeneous one-to-one conjugation products, available in virtually unlimited amount. Various assays were developed using this strategy: a short review of the most interesting applications is presented, as well as the cloning, purification and initial characterization of an endothelin-1-aequorin conjugate suitable for developing a competitive immunoassay for endothelin-1, a potent vasoconstrictor peptide, involved in hypertension.
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