Single Protein Nanobiosensor. Grid Array (SPOT-NOSED)
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1 Single Protein Nanobiosensor Grid Array (SPOT-NOSED)
2 Single Protein Nanobiosensor! Abstract Recent advances in bio- and nano-technology have opened the possibility to develop bio-electronic sensors based on the properties of single biomolecules. They will be the ultimate limit in miniaturization, specificity and sensitivity and the closest bio-electronic mimic of animal sensing systems. This project explores the possibility to develop an olfactory nanobiosensor array based on electrical properties of single olfactory receptors. In vivo odorant detection odorant +olfactory receptor In vitro odorant detection odorant +olfactory receptor signal transduction by enzymatic cascade electrical signal detection and processing electrical signal to brain odorant identification odorant identification
3 In vivo odorant identification! Anatomy of the mammal olfactory system: Fig. A Olfactory Neuroepitelium The sensing cells are olfactory sensory neurons (OSN) in the upper reaches of the nasal cavity. From one end, they reaches the tissue surface with cilia; from the other end, an axon projects directly to higher brain regions. Fig. B OSN Membrane At the olfactory cilia in the nasal mucus, a cascade of enzymatic activity transduces the binding of an odorant molecule to the receptor into an electrical signal towards the brain OR odorant B Fig. C Olfactory Receptor Olfactory receptors (Ors) are proteins belonging to the GPCR family. A schematic view of the proposed 3D structure is shown with seven transmembrane regions connected by intra- and extra-cellular loops. A C
4 In vitro odorant identification! Towards single protein bio-sensors: 1. Production of Olfactory Receptor (OR)! From natural sources and from expression! ORs are refractory to high yield expression! Test GPCRs proteins - bovine rhodopsin (photoreceptor) - rat somatostatin (neurotransmitter receptor) 2. AFM nanotools and nanoelectrodes fabrication AFM cantilever OR NH Metal 2 Tip 3. Transfer of the OR onto conducting substrates! OR into a Langmuir-Blodgett film! Transfer of OR films onto functionalized substrates by Langmuir-Blodgett technique COOH Nanoelectrodes lipidic bilayer antibody 4. Electrical characterisation of the transferred OR OR Rhodopsin 2D model 25nm gold nanoelectrodes gold substrate
5 Electrical Characterisation of Single Protein (ORs) Activity! AFM (Atomic Force Microscope) measurements Develop and fabrication of specific front-end circuitry to probe single olfactory receptor activity by: I-V measurements Impedance spectroscopy Capacitance measurements Noise measurements Main objective: tracking of very low current variations T i [A] i Odorant time [s] persistance Expected waveform of the current signal from the olfactory receptor when binding to the odorant. In order to correctly measure the signal, a fast-response preamplifier is needed. conductive cantilever I OR V substrate functionalisation self-assembled monolayer (SAM) Current I + applied voltage V conductive substrate Amplifier Specifications: Low noise High gain High bandwidth
![Capacitance measurements Noise measurements Main objective: tracking of very low current variations T i [A] i Odorant time [s] persistance Expected waveform of the current signal from the](/docs-images/57/6052752/images/page_5.jpg "olfactory receptor when binding to the odorant. In order to correctly measure the signal, a fast-response preamplifier is needed.")
6 Electrical Characterisation of Single Protein (ORs) Activity! Low-noise wide-bandwidth amplifier " Integrator-differentiator scheme - Minimum noise - Large bandwidth " New active feedback - Separate path for DC leakage current - No feedback-capacitor reset circuitry i inp ut R F I DC Integrator C p C F i signal Active Feedback C D Differentiator R D AC v out " Application: continuous DC and AC measurement " Features: CDRD - AC gain: G = C independent from R F F - bandwidth: independent from R F, BW [1Hz, 1MHz] - noise: same as standard AFM amplifier i 4KT in + 4π eop F + RF 2 2 ( C C ) f P Gain Integrator Differentiator 1MHz logf
7 Electrical Characterisation of Single Protein (ORs) Activity! Low-noise wide-bandwidth amplifier * (AFM-compatible) Input in a separate circuit front-end DC out AC out AFM system (topview) 9mm Front-end mounted into the AFM head * Patent n. MI2003A mm
8 Electrical Characterisation of Single Protein (Ors) Activity! Electronic instrumentations under development R For nanoelectrodes - after using AFM, measurements will be performed by nanoelectrodes AC DC ~ Amp. Vout - the new low-noise large-bandwidth Nanoelectrodes setup amplifier will be optimised and implemented for nanoelectrodes setup GND No constraints in circuit size improved mounting Reduction of parasitics Optimized choice of components For noise measurements - Detection of odorant binding Amplifier Frequency selector - Need to avoid instrument noise a custom-made Correlation Spectrum Analyzer will be fully designed and built Input DUT Amplifier B B Frequency selector Averager Output Block diagram of the Correlation Spectrum Analyser
9 Research Partners Politecnico di Milano (Italy) Istituto Nazionale per la Fisica della materia (Italy) Universitat de Barcellona (Spain) Laboratoire de biologie cellulaire et moléculaire (France) Centro Nacional de Microelectrónica (Barcellona,Spain) Laboratoire d ingénierie et de fonctionnarisation de surface (École Centrale de Lyon, France) IST SPOT-NOSED Project Funded by the Future and Emerging Technologies arm of the IST Programme FET-Open scheme.
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