New advances in optical biosensing

Transcription

1 New advances in optical biosensing Francesco Baldini Chemical and Biochemical Optical Sensor Group Institute of Applied Physics, National Research Council Florence Italy

2 The optical solution in biosensing Optical biochip for sepsis diagnosis in POCT applications Nanosensing with nanostructured materials for the monitoring of intracellular parameters

3 POCT applications need of physicians to have a fast response to formulate the right diagnosis or to decide the correct therapy, avoiding to deliver the samples to the central laboratories and to wait for the results of the analysis Multianalyte detection (a few biomarkers, differently from genomics or proteomics) Cardiac markers: troponin, myoglobin, D-dimer, brain-type natriuretic peptide, creatine kinase-mb Sepsis: C-reactive protein, procalcitonin, neopterin, tumour necrosis factor α, myeloperoxidase, interleukine-6, interleukine-8 and interleukine-10 need of discrimination between viral and bacterial inflammation / sepsis in intensive care patients, or in any case need of a fast identification of the origin of infections Every hour delay until administration of an effective calculated antibiotic treatment in septic shock increases mortality by 7%!

4 Biosensor microarray Microarray system consists of array of probes on a substrate (chip) Optical biochip label-based systems, in which the optical signal that depends on the analyte investigated, comes from a label with suitable optical properties (mainly fluorescence or chemiluminescence) label-free systems, in which the interaction analyte/sensitive layer gives rise to a modification of the optical signal due to a change in the refractive index of the layer deposited on the substrate (interferometry, surface plasmon resonance) in clinical applications in which the sample matrix can be very complex (as in the case of whole blood or plasma) the use of a label-based system may be preferable

5 Fluorescence anisotropy electric dipoles exhibit anisotropic emission when the distance from a dielectric interface is small or comparable with the emitted wavelength Mac Craith et al, Electronic Letters, 41(12), , 2005

6 The optical chip based on fluorescence anisotropy F. Baldini et al., Anal. Bioanal. Chem., 391, pp , 2008

7 Geometrical configuration for multiassay

8 Optical chip produced by injection moulding with thirteen channels

9 Sandwich assay Analyte Capture mab Detection mab Label CRP clone C5 clone C7 DY647 PCT PROC1 3G3 PROC4 6C6 λ exc = 635 nm λ em = 675 nm

10 CRP assay PCT assay F.Baldini et al., Anal. Bioanal. Chem., 393, 1183, 2009 F.Baldini et al., Sens. Actuat. B, 139, 64, 2009

11 Portable prototype

12 Intensity Chip-scanning Measure Zero Channels CRP assay: channel 2-6 PCT assay: channel 8-13

13 Nanosensing in cells Combination of photonics and nanotechnology Nanovectors of sensing molecules immobilised on nanoparticles Passive delivery NP modification to make them hydrophilic Facilitated delivery immobilisation on the NP surface of molecules recognised by the cell (e.g. peptide, protein) capable to facilitate the initial NP cell membrane interaction Active delivery direct manipulation of the cell (microinjection and electroporation)

14 Optical beacons Design, development and characterisation of nanostructured materials capable to vehicle nanocarriers of molecules in cells acting as drug and sensor at the same time Nanocarriers: Carbon nanotubes, PMMA nanoparticles, gold nanoparticles, silica nanoparticles Drug/sensor: Antisense oligonucleotides designed as molecular beacons capable to emit fluorescence after the hybridization with the complementary sequence Selected antisense oligonucleotides: specific for the mrna coding the antiapoptotic proteins survivin and Bcl-2

15 Design of the molecular probes for survivin 5'ATTO 647-GAGAAAGGGCTGCCATTCTC-3'BBQ Atto 647N Blackberry 650

16 MB calibration curve with the synthetic target 5'-GAGAATGGCAGCCCTTTCTC-3'

17 MB calibration curve with the synthetic target 5'-GAGAATGGCAGCCCTTTCTC-3'

18 ph nanosensing Fluoresceine immobilization on carbon nanotubes The ether spacers have a twofold function: i) to separate the dye from the nanotube in order to decrease the fluorescence quenching; ii) to provide a better water solubility of the final product.

19 Amount of fluorescein by thermo gravimetric assay Fluorescein / grams of modified MWCNT MW-Fluo1 78mg MW-Fluo2 117mg MW-Fluo3 39mg Such a result is approximately consistent with 1 fluorescein group every 399 (MW- Fluo1), 401 (MW-Fluo2) and 1794 (MW-Fluo3) carbon atoms, respectively

20 Emission Optical charaterisation integrals integrals MWF2 DoseResp Fit of Sheet1 C ,9 6,9 6,4 5,9 5,4 4,8 4,4 3,8 3,4 2,9 2,5 2, Model Equation Reduced Chi-Sq r DoseResp y = A1 + (A2-A1 )/(1 + 10^((LOG x0-x)*p)) 985,00473 Adj. R-Square 0,99858 Value Standard Error C A1 711, ,38189 C A2 3468, ,72884 C LOGx0 6, ,04735 C p 0, ,03157 C span 2757,23672 C EC ,04818 C EC50 2,658E6 C EC80 2,44128E Wavelength ph Fluorescence spectra of MW-Fluo2 ph calibration curve next step vehiculation in cell culture F. Baldini et al., Proc. SPIE Vol. 7574, 75740C-75740C-8 (2010)

21 Conclusion POCT device for sepsis diagnosis implementation of a novel optical platform development of CRP and PCT sandwich assays first tests on patient samples Optical nanosensing design and characterization of molecular beacons in solution design, realisation and characterisation of ph nanosensors on CNT

22 Chemical and Biochemical Optical Sensor Group - Institute of Applied Physics Francesco Baldini Giacomo Ghini Ambra Giannetti Francesco Chiavaioli Cosimo Trono Folco Senesi Collaborations - Institute of Chemistry of Organometallic Compounds ICCOM - CNR- Florence, Italy - Dept Pharmacology - University of Pisa, Italy - Istituto Toscano Tumori ITT Florence, Italy - Institute of Complex Systems ISC- CNR Florence, Italy - Datamed - Milano Italy - Institut für Physikalische und Theoretische Chemie, Tübingen, Germany - EXBIO Praha, Czech Republic - Helmholtz Zentrum München, Institute of Ecological Chemistry, Neuherberg, Germany - Veterinary Research Institute, Brno, Czech Republic Funds - European IP project, CARE-MAN - HealthCARE by Biosensor Measurements and Networking ( ) - Tuscany Regional project NANOCELL Optical Nanosensing in cell ( )

23 CONVEGNO NAZIONALE SENSORI Innovazione, attualità e prospettive Roma Febbraio 2012 Società Italiana di Ottica e Fotonica (SIOF) Società di Chimica Italiana (SCI) Associazione Italiana Sensori e Microsistemi (AISEM) Associazione italiana Ambient Assisted Living (AitAAL) Associazione Italiana Misure Elettriche ed Elettroniche (GMEE) Associazione per la Tecnologia dell Informazione e delle Comunicazioni (AICT) Società Italiana di Fotobiologia Società Italiana di Biofisica Pura e Applicata (SIBPA)