Robust biomolecules as advanced tools for biotech applications - Nano-Innovation 2018, Rome, Italy - September 12, 2018
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Robust biomolecules as advanced tools for
biotech applications
Dr. Sabato D’Auria
Laboratory for Molecular Sensing
National Research Council
Avellino, Italy
Nano-Innovation 2018, Rome, Italy - September 12, 2018
Recognition Element Signal Transducer
Chemical Probe Optical
Enzyme Electro-Chemistry
Protein Calorimetric
Antibody
analytes
Recognition element
transducer
amplifier
microelectronics
Advantages of Protein Fluorescence Sensors - Specificity - Reversibility - Wide range of affinity constants - Low-cost sensors - Water soluble - Wide range of analytes - Easy to mutate and express in hosts - Enzymes can be labeled with extrinsic fluorophores - Optical properties are not linked to the specificity
Main concerns on the utilization of enzymes as fluorescence biosensors 1)- Stability 2)- Enzyme activity 3)- Analyte consumption
Temperature over 100 oC
pH below 1
Far-UV circular dichroism spectra from 20 oC – 95 oC
Thermostable proteins as long-term stability probes: example of glucose sensing by mesophilic and thermophilic glucokinase
Biological interactions represent the base for a specific sensing strategy
Fluorescence Platform for advanced sensing methodologies
Light Source Target Analyte
Intensity Intensity ratio Time Phase Modulation
DF
Intensity
A B Slope-1/t Dm
Wavelenght Wavelenght Time Time
Dm DF
DF or Dm
Intensity
A/B
t
[Analyte] [Analyte] [Analyte] [Analyte]Fluorescence Correlation Spectroscopy / Single Molecule
Detection
11Multi-analyte assay by nano-patterning biomolecules specifically labeled
Near-IR Fluorescence Sensing for Assays in Real Matrices and Whole Blood
Li-Cor Inc.
Info ConfidentialLipocalin Scaffold: Manipulate them for sensitivity and specificity improvements
Reverse docking results. The graphical output provided by IdTarget .
Capo A, Pennacchio A, Varriale A, D'Auria S, Staiano M (2018) The porcine odorant-binding protein as molecular probe for benzene detection. PLoS ONE 13(9): e0202630.
RET-BASED ASSAY ON CARBON NANOTUBES FOR
MONITORING ODORANT MOLECULES
3,0
Fluorescence Intensity (a.u.)
OBP Alone
OBP with Antrhracene
OBP with Anthracene and Octene Tri-ol
2,5
2,0
1,5
1,0
0,5
0,0
300 350 400 450 500 550 600
Wavelength (nm)Diphenylamine – Dimethylftalate- DinitroToluene- Resorcinolo
In collaborazione con Prof. Luigi Zeni, Seconda Università di Napoli
Plastic Optical fiber as an innovative material for sensing applications With Prof. Luigi Zeni, Seconda Università di Napoli
Biophotonic Ring Resonator for Ultrasensitive Detection of Simulant Organophosphorus Agents Prof. Andrea Melloni Politecnico di Milano This work is supported by the NANOCAP project A-1084-RT-GC that is coordinated by European Defence Agency and funded by 11 contributing members (Cyprus, France, Germany, Greece, Hungary, Italy, Norway, Poland, Slovakia, Slovenia, and Spain)
High Technology from Lab to Street, that is offering the
opportunity to do-it-yourself analyses
From lab...
... to street
Dr. Sabato D’AuriaMetal-induced Fluorescence enhancement
Metal-enhanced fluorescence
Unlabelled DNA DAPI- and IP-
labelled DNA
Increase
in RET
effect
Dr. Sabato D’AuriaMetal-enhanced fluorescence & TIRF excitation Dr. Sabato D’Auria
Silver islands boost the fluorescence signal Dr. Sabato D’Auria
……and TIRF makes no washing steps needed
with washing no washing
600
fluorescence at 590 nm, a.u.
500
400
300
200
100
0
TIR mode - TIR mode - FF mode - FF mode -
SIF glass SIF glass
Dr. Sabato D’Auria……and the device is ready
Sample
50n
M
5 nM
500n
M
Dr. Sabato D’AuriaTAKE HOME MESSAGE
In conclusion, putting together the effect of silver island
with TIRF excitation represents the way to move from
lab complex instrumentations to the on-the-site analyses
that could be potentially performed at home.
Dr. Sabato D’AuriaCeliSens will produce a user-friendly portable (~10 cm) devices
for quantification of gluten traces in the food.
Device Core
Nuovo sistema portatile per la rilevazione di tracce di glutine negli alimenti
D’Auria S., et al.
Brevetto Nazionale (2013) n. TO2013A001057;Device Design
Acquisition chamber
Laser diode &
Operating &
Focusing
Acquisition
optics
Open Source
software
Hardware
controller chassis
Photodiode
Array detector
Arduino UNO
Electric wire sketch Programmable
Digital Output controller
Signal3D Printable Sensing Device with
ElectronicsPortable Device
Personnel D’Auria Sabato dott.ssa Staiano Maria dott. Varriale Antonio dott.ssa Camarca Alessandra dott. Ausili Alessio dott.ssa Pennacchio Angela dott. Capo Alessandro dott.ssa Majoli Adelia dott.ssa Capacchione Clotilde dott. Santorelli Alessandro
Funded Projects EU FP7 KKBE - MARINE environmental in situ Assessment and monitoring tool BOX” – 2014-2018 Grant Agreement Number 247868 EU FP7 Security CUSTOM Drugs And PreCUrsor Sensing By ComplemenTing Low COst Multiple Techniques 2009-2013 Grant Agreement Number 242387 BIOsensors for point detection based on nanostructured opTical components for quick deploYment in an overall CBRN EuroPEan operational capability Grant Agreement Number A-1152-RT-GP Novel Nanostructured optical components for CNBR detection and high performance opto-microwaves links Grant Agreement Number A-1084-RT-GC Flagship Project – NanoMAX Design and realization of a nanotechnology-based assay for an early detection of markers for endocrine tumors A New glucometer based-assay for detection of toxins in milk Conoscenze Integrate per Sostenibilità e Innovazione del Made in Italy Agroalimentare CAMPUS - Qualità delle produzioni tipiche campane ed il suo territorio: approcci innovativi ed integrati per rafforzare la competitività del sistema Agroalimentare” – QUARC
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