Single Protein Nanobiosensor Grid Array (SPOT-NOSED)home.deib.polimi.it/sampietr/Spotnosed/spotnosedsummary.pdfPolitecnico di Milano - Dipartimento di Elettronica OR odorant A C B

Politecnico di Milano - Dipartimento di Elettronica

Single Protein Nanobiosensor

Grid Array

(SPOT-NOSED)


Single Protein Nanobiosensor

! AbstractRecent advances in bio- and nano-technology have opened the possibility to developbio-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 In vitro odorant detection

odorant identification

odorant +olfactory receptor

signal transductionby enzymatic cascade

electrical signal to brain

odorant identification

odorant +olfactory receptor

electrical signal detectionand processing


OR

odorant

A

C

B

In vivo odorant identification

! Anatomy of the mammal olfactory system:

Fig. A Olfactory NeuroepiteliumThe sensing cells are olfactory sensory neurons (OSN) in the upper reaches of the nasal cavity. From one end, they reaches the tissue surface with 20-30 cilia; from the other end, an axon projects directly to higher brain regions.

Fig. B OSN MembraneAt 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

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 transmembraneregions connected by intra- and extra-cellular loops.


In vitro odorant identification

! Towards single protein bio-sensors:

1. Production of Olfactory Receptor (OR)

2. AFM nanotools and nanoelectrodes fabrication

3. Transfer of the OR onto conducting substrates

4. Electrical characterisation of the transferred OR

! From natural sources and from expression! ORs are refractory to high yield expression! Test GPCRs proteins

- bovine rhodopsin (photoreceptor)- rat somatostatin (neurotransmitter receptor)

OR

Metal Tip

AFM cantilever

COOH

NH2

Nanoelectrodes

25nm goldnanoelectrodes

gold substrate

OR

antibody

lipidic bilayer

Rhodopsin2D model

! OR into a Langmuir-Blodgett film! Transfer of OR films onto functionalized substrates

by Langmuir-Blodgett technique


Electrical Characterisationof Single Protein (ORs) Activity

! AFM (Atomic Force Microscope) measurements

• I-V measurements• Impedance spectroscopy• Capacitance measurements

• Noise measurements

− Develop and fabrication of specific front-end circuitry to probe singleolfactory receptor activity by:

time [s]

i [A] T

∆i

Odorant 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.

Amplifier Specifications:• Low noise• High gain• High bandwidth

− Main objective:tracking of very low current variations substrate

functionalisationself-assembled

monolayer (SAM)

−

conductive cantilever

conductivesubstrate

applied voltage

V

Current I

VI+

OR



! Low-noise wide-bandwidth amplifier

logf

Gain

IntegratorDifferentiator

1MHz

" Integrator-differentiator scheme- Minimum noise- Large bandwidth

" New active feedback- Separate path for DC leakage current- No feedback-capacitor reset circuitry

" Application: continuous DC and AC measurement

" Features:- AC gain: independent from RF

- bandwidth: independent from RF, BW ≈ [1Hz , 1MHz]

- noise: same as standard AFM amplifier

F

DD

CRCG =

( ) 22222 44 fCCeRKTi PFop

Fin ++≅ π

AC vout

iinp

ut

CF

CDCpRF

RDisignal

ActiveFeedback

IDC

Integrator Differentiator



! Low-noise wide-bandwidth amplifier * (AFM-compatible)

front-end

in a separatecircuit

ACout

8mm

9mm

AFM system(topview)

Front-end mounted into the AFM head

Input

* Patent n. MI2003A002543

DCout


Electrical Characterisationof Single Protein (Ors) Activity

! Electronic instrumentations under development

~AC

DC

GND

VoutAmp.

R

~AC

DC

GND

VoutAmp.

R

~AC

DC

GND

VoutAmp.

R

Nanoelectrodes setup

• No constraints in circuit size• improved mounting• Reduction of parasitics• Optimized choice of components

DU

TB

B

Input

Frequency selector

Frequency selector

Ave

rage

r

Amplifier

Output

Amplifier

− For noise measurements- Detection of odorant binding- Need to avoid instrument noise

a custom-made Correlation Spectrum Analyzer will be fully designed and built

Block diagramof the Correlation

Spectrum Analyser

− For nanoelectrodes- after using AFM, measurements will be

performed by nanoelectrodes

- the new low-noise large-bandwidth amplifier will be optimised andimplemented for nanoelectrodes setup


Research Partners

Politecnico di Milano (Italy)

Laboratoire de biologie cellulaire et moléculaire

(France)

Laboratoire d’ingénierie et de fonctionnarisation de surface

(École Centrale de Lyon, France)

Istituto Nazionale per la Fisica della materia

(Italy)

Universitat de Barcellona (Spain)

Centro Nacional de Microelectrónica

(Barcellona,Spain)

IST-2001-38899-SPOT-NOSEDProject Funded by the Future and Emerging Technologies

arm of the IST Programme FET-Open scheme.

Single Protein Nanobiosensor Grid Array (SPOT-NOSED)home.deib.polimi.it/sampietr/Spotnosed/spotnosedsummary.pdfPolitecnico di Milano - Dipartimento di Elettronica OR odorant A C B

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