At NANOTEC Institute, several research activities are in progress concerning the optimization of a wide range of biosensors able to respond, alone or integrated in multifunctional sensing platform, to specific needs of several biological applications. On one side, we employ different biorecognition elements (antibodies, aptamers or molecular imprinted polymers) to achieve specificity. On the other, we exploit various transduction methods ranging from optical to electrical and mechanical ones in order to miniaturize the assay, reduce the cost and/or increase the sensitivity. Moreover, our studies investigate also the use of biomaterials for electronics applications. More in detail:
Biorecognition elements for sensor specificity
Many biological events are closely associated with the specific binding between biomolecules that complement each other in shape, size, charge and chemical functionality, and self-organize with nanoscale dimension at an interface (i.e., lipid assemblies, proteins, viruses). To attain high specificity and efficiency, biosensors exploit molecular recognition such as DNA base pairing or antigen-antibody reactions but we also employ aptamers and molecular imprinted polymers as emerging molecular probes. However, achieving rapid and efficient specific molecular interaction often depends on immobilization of receptors to solid surfaces with appropriate surface density, preserving the functional conformation and optimizing the presentation of the binding fragments towards the target analyte. Moreover, the ability to bind/release a molecule of interest in response to an external stimulus (i.e., temperature, pH, irradiation), has a practical impact in the field of bioseparations (purification of proteins, enzymes, pharmaceuticals) and drug delivery. In this respect, in situ analytic techniques able to determine the absolute orientation and conformation of bio- and biomimetic molecules at the interfaces are of paramount relevance. Furthermore, dynamically probing the active sites during the binding/releasing event would provide a molecular-level understanding of the receptor-analyte interaction, and eventually shed light on how the performance of biological processes in living organisms depends on the biomolecular interfacial architectures. Sum-frequency generation vibrational spectroscopy (SFG-VS) allows achieving the conformational structure and orientation distribution of biomimetic and bio- molecules at interfaces, in the working environment for chemical and biochemical sensing. As a second-order nonlinear optical processes, SFG-VS has been proven to be a versatile tool for non-invasive probing of any interface accessible by light, with intrinsic surface specificity, chemical selectivity and sub-monolayer sensitivity. Being electric-dipole forbidden in centrosymmetric bulk media but allowed at interfaces where inversion symmetry is naturally broken, SFG is highly interface-specific. SFG is a process in which two input laser beams at VIS and IR frequencies interact in a medium and generate an output beam at frequency ω_SFG=ω_VIS+ω_IR. When ω_VIS (ω_IR) and/or ω_SFG are tuned over resonances, SFG is resonantly enhanced thus yielding surface spectroscopic information. For molecular systems, vibrational spectroscopy is often more selective, since it permits identification of molecular species and provides information about their functional groups. Therefore SFG-VS, where the input beam ω_IR is scanned over vibrational resonances, has been largely exploited as surface-specific analytical probe for molecules at interfaces. SFG-VS is sensitive to the average polar orientation of each moiety and therefore to the overall molecular conformation. As a coherent process, the output beam is highly directional, allowing for in situ non-invasive remote sensing of the interface.
Optical read-out: Sum-frequency generation vibrational spectroscopy (SFG-VS) and plasmonics
Optical sensor technology offers significant opportunities in the field of medical research and clinical diagnostics, spanning from colorimetric and fluorescent assays to more advanced approaches particularly suitable for the detection of a few molecules in highly-diluted solutions. At CNR-NANOTEC, several methods are under investigation. For example, a colorimetric “point-of-care” device was optimized exploiting electrochromism for the detection of ions in biological fluids. For few molecules detection, we are then working on label-free plasmonic biosensors based on metallic nanostructures and metamaterials. To detect lower molecular weight (<500 Da) biomolecules in highly-diluted solutions, we are investigating a hyperbolic metamaterial (HMM)-based plasmonic biosensor platform, which can support highly confined bulk plasmon guided modes in a wide wavelength range, from visible to near infrared. Hyperbolic metamaterials are perhaps the most unusual electromagnetic metamaterials, featuring hyperbolic (or indefinite) dispersion because one of their principal components has the opposite sign to the other two. Our HMM sensor device shows many highly sensitive resonant modes with a maximum sensitivity of 30,000 nm/RIU and a maximum figure of merit of 590 at near IR wavelength. In collaboration with CNR-IMM, we have also recently optimized colloidal nano-lithography as a cheap approach to fabricate planar distributions of plasmonic nanostructures with tailored optical functionalities.
Mechanical read-out: QCM and SAW
Another trasduction mechanism under investigation at CNR-NANOTEC is based on a mechanical approach. If opportunely functionalized, a quartz crystal microbalance (QCM) interacts with analytes in solution to estimate the concentration or can be used for cell studies. In our labs, we have employed QCM to study peptide-antibody interactions to allow detection of new biomarkers for early diagnosis of aggressive forms of cancer. Another application was in the field of ophthalmology to search for innovative coatings to improve contact lens performances and produce biofouling surfaces. Recently QCM was used to investigate cancer cell-drug interactions and to probe aptamer-toxin interactions in the perspective of developing biosensors for monitoring food quality. To further improve the sensitivity in mass detection, we are also working on Surface Acoustic Wave (SAW) technology and we recently optimized both delay lines and resonators.
Electrical read-out: E.I.S. and MR biosensors
Tailoring low cost, portable, easy handled devices for simple and rapid assays is a major goal for current technology research. In this respect, an electrical read-out can provide advantages. In our labs, we optimized electrochemical impedance spectroscopy (EIS) based biosensors consisting of couples of interdigitated electrodes made of gold on a glass substrate integrated with a microfluidic module for the automatic handle and delivery of solutions and samples to the sensing areas of the device. This EIS platform has been used in Lecce for diagnostics, food control, environmental monitoring and cell studies.
Recently, in Bari, a label-free impedance device was also optimized for virus early detection in apparently asimptomatic plants at attomolar concentration in collaboration with the CNR IPSP – Istituto per la Protezione Sostenibile delle Piante, UoS Bari. Specifically, Tomato the mosaic virus (ToMV) and the Turnip yellow mosaic virus (TyMV) were analyzed and the different virus charges have been found to regulate the impedance response and the electrochemical interaction with a textured surface, enabling selectiveness and quantification.
In this frame, properly functionalized nanostructures can act as high sensitive recognition element due to the high surface to volume ratio. As a different strategy, in our labs, in collaboration with Technion Institute in Haifa (Israel), functionalized silicon nanowires arrays (SiNWs) were embedded on interdigitated electrodes (IDEs) for TFT devices responding to low Volatile Organic Compounds (VOCs) concentration produced by human breath when affected by gastric cancer for a novel non invasive cancer diagnostic technique.
At NANOTEC we are also investigating magnetoresistance sensors as transducers since they promise excellent sensitivity in the detection of biomolecules labeled by magnetic particles. A new H2020 project will exploit this technology for achieving early detection of neurodegenerative diseases (Alzheimer and Parkinson).
Plasma processing of materials for sensing
Plasma processing can be used to modify the surface properties of materials for sensing. Plasma assisted functionalization of surfaces can be performed in order to introduce nitrogen and oxygen containing groups other than micro-nanopatterned surfaces and promote further immobilization or analysis of molecules
In bio-molecules, like amino-acid derivatives, proteins etc.. the water bounding is of fundamental importance. This feature is generally encountered in living cells, where water binds to the structure and fluctuates from an ordered to a disordered structure. In this respect melanin and melanin-like materials have displayed a peculiar hydration dependent modification of their properties and specifically of the electrical one, leading to consider melanin as an electret able to store water derived charges.
Even if such behavior was a well known feature disclosed in the past, only recently our group was able to demonstrate that it is possible not only to relate the amount of the stored charge to the magnitude of an applied signal (e.g. a continuous voltage) but also that the transmitted information is not erasable and can be stored for a long time. Moreover, depending from the hydration state, the origin of the storage is switching from an electron/hole charge trapping to a polarization effect, the latter regulated by the residual water ions. A recently afforded fundamental issue concerns the kind of physicochemical interactions between water molecules and eumelanin in determining the functionalities, which possibly can hinder the development of rational strategies to fine tune the ionic-electronic conduction mechanisms, a primary goal in the design of bio-electronic devices useful for memory recovering or powering.
As valid alternative to melanins-based biological system, Polydopamine (pDA) constitutes an attractive platform due to the easy deposition by dip-coating, the eumelanin-like properties and the possibility to tune its properties by binding to proper functionalities. As an example, our group thanks to the collaboration with the Prof. M. d’Ischia’s group (Chemistry Dpt at the University of Naples) was able to demonstrate the feasibility of chemical doping and tuning of the electrical transport when pDA is co-polymerized with electron-donating systems. Inspired by the powerful photosensitizing properties of the red hair pigments pheomelanins, a photoresponsive cysteine-containing polydopamine obtained via oxidative copolymerization of dopamine (DA) and 5-S-cysteinyldopamine (CDA) in variable ratios was produced and tested. In particular, the obtained copolymer replicates in a certain extent, the structure of neuromelanin. The use of the p(DA/CDA) copolymer constituted in this case a first example of technological exploitation of photoactive, red hair-inspired biomaterials as soft enhancement layer for a bio-friendly bio- device with a chemically tunable response to visible light. Interestingly, respect to pDA, the p(DA/CDA) copolymers displayed an interesting properties binding water in a fashion similar to those of biological materials and a marked photoimpedance response to light stimuli.
- M. Bianco, A. Sonato, A. De Girolamo, M. Pascale, F. Romanato, R. Rinaldi, V. Arima An aptamer-based SPR-polarization platform for high sensitive OTA detection Sensors & Actuators: B. Chemical 314-320 (2017) ISSN: 0925-4005; doi: 10.1016/j.snb.2016.10.056
- A. Aprile, F. Ciuchi, R. Pinalli, E. Dalcanale, P. Pagliusi, Probing Molecular Recognition at the Solid-Gas Interface by Sum-Frequency Vibrational Spectroscopy, Journal of Physical Chemistry Letters, 7, 3022-3026, (2016) ISSN 1948-7185; doi: 10.1021/acs.jpclett.6b01300
- A. Aprile, P. Pagliusi, F. Ciuchi, R. Pinalli, E. Dalcanale, Probing cavitand-organosilane hybrid bilayers via sum frequency vibrational spectroscopy, Langmuir 30, 12843 (2014) ISSN: 0743-7463; doi: 10.1021/la503150z.
- S. R. Cicco, M.Ambrico, P.F.Ambrico, M.Mastropasqua Talamo, A.Cardone,T.Ligonzo, R. DiMundo, C. Giannini, T. Sibillano, G.M. Farinola, P.Manini,A.Napolitano, V. Criscuolo, M. D’Ischia A water-soluble eumelanin polymer with typical polyelectrolyte behaviour by triethyleneglycol N-functionalization, Journal of Material Chemistry, C 3, 2810-2816 (2015) ISSN: 2050-7526; doi: 10.1039/c4tc01997k
- M. Ambrico, P.F. Ambrico, A. Cardone, S.R. Cicco, F. Palumbo, T. Ligonzo, R. di Mundo, V. Petta, V. Augelli, P. Favia and G. M. Farinola Melanin-like polymer layered on a nanotextured silicon surface for a hybrid biomimetic interface, Journal of Material Chemistry C, 2,573, (2014) ISSN 2050-7534; doi: 10.1039/c3tc31327a
- M. Ambrico, N.F. Della Vecchia, P.F. Ambrico, A. Cardone, S.R.Cicco, T. Ligonzo, R.Avolio and A. Napolitano, A Photoresponsive Red-Hair-Inspired Polydopamine Based Copolymer for Hybrid Photocapacitive Sensors, Advanced Functional Materials, 24,7161-7172, (2014), ISSN: 1616-3028; doi: 10.1002/adfm.201401377
- N. F. Della Vecchia, R. Marega, M. Ambrico, M. Iacomino, R. Micillo, A. Napolitano, D. Bonifazi and M. d’Ischia Tailoring melanins for bioelectronics: polycysteinyldopamine as an ion conducting redox-responsive polydopamine variant for pro-oxidant thin films, Journal of Material Chemistry C, 3,6525-6531,(2015) ISSN 2050-7534; doi: 10.1039/c5tc00672d
- M. Ambrico (Lead Guest Editor) Special issue: Melanin, a long lasting history bridging natural pigments and organic bioelectronics, Polymer International, 65,11 (2016) ISSN: 1097-0126; doi: 10.1002/pi.5239
- G. Da Ponte, E. Sardella, F. Fanelli, S. Paulussen, P. Favia. Atmospheric pressure plasma deposition of poly lactic acid-like coatings with embedded elastin. Plasma Processes and Polymers 11-4, 342-352 (2014) ISSN: 1612-8850; doi: 10.1002/ppap.201300130
- M. Bianco, V. Guarino, G. Maruccio, G. Galli, E. Martinelli, G. Montani, R. Rinaldi and V. Arima Non-Biofouling Fluorinated Block Copolymer Coatings for Contact Lenses Sci. Adv. Mater. 7, 1387-1394 (2015). ISSN: 1947-2935; doi: 10.1166/sam.2015.2056
- Z. Ameer, E. Primiceri, F. De Feo, M. S. Chiriacò, A. G. Monteduro, G. Maruccio and R. Rinaldi, DNA sensors with impedimetric and magnetoresistive transduction – A comparison study, Proceedings of 2014 11th International Bhurban Conference on Applied Sciences and Technology, IBCAST 2014 65-68 (2014). ISSN: 2151-1403; doi: 10.1109/IBCAST.2014.6778122
- S. Chiriacò, F. de Feo, E. Primiceri, A. G. Monteduro, G. E. de Benedetto, A. Pennetta, R. Rinaldi and G. Maruccio, Portable gliadin-immunochip for contamination control on the food production chain Talanta 142, 57-63 (2015) ISSN: 00399140; doi: 10.1016/j.talanta.2015.04.040
- A. Colombelli, M. G. Manera, R. Rella, S. Rizzato, E. Primiceri, A. G. Monteduro and G. Maruccio, Colloidal lithography fabrication of tunable plasmonic nanostructures, IET Conference Publications 2015, Vol. 2015. doi: 10.1049/cp.2015.0148
- S. Chiriacò, E. Primiceri, F. De Feo, A. Montanaro, A. G. Monteduro, A. Tinelli, M. Megha, D. Carati and G. Maruccio, Simultaneous detection of multiple lower genital tract pathogens by an impedimetric immunochip, Biosensors and Bioelectronics 79, 9-14 (2016) ISSN: 0956-566; doi: 10.1016/j.bios.2015.11.100
- V. De Matteis, A. Cannavale, L. Blasi, A. Quarta, G. Gigli Chromogenic device for cystic fibrosis precocious diagnosis: A “point of care” tool for sweat test Sensors and Actuators B: Chemical 225, 474–480 (2016) ISSN: 09254005; doi: 10.1016/j.snb.2015.11.080
- G. Palazzo , D. De Tullio, M. Magliulo , A. Mallardi , F. Intranuovo , M.Y.Mulla , P.Favia, I.V.-Lundin , L. Torsi, Detection Beyond Debye’s Length with an Electrolyte-Gated Organic Field-Effect Transistor, Adv. Mater. 27, 911–916 (2015) ISSN: 0935-9648; doi: 10.1002/adma.201403541
- V. Sreekanth, Y. Alapan, M. ElKabbash, U. A. Gurkan, E. Ilker, M. Hinczevski, A. De Luca and G. Strangi, Extreme sensitivity biosensing platform based on hyperbolic metamaterials Nature Materials 15 (6), 621 (2016) ISSN: 1476-1122, DOI: 10.1038/NMAT4609
- V. Caligiuri, R. Dhama, K. Valiyaveedu Sreekanth, G. Strangi and A. De Luca Dielectric singularity in HMM: the inversion point of coexisting anisotropies, Scientific Reports (Nature Publishing Group) 6, 20002 (2016) ISSN: 2045-2322; doi: 10.1038/srep20002
- V. Sreekanth, K. Hari Krishna, A. De Luca and Giuseppe Strangi Large spontaneous emission rate enhancement in grating coupled hyperbolic metamaterials Scientific Reports (Nature Publishing Group) 4, 6340 (2014) ISSN: 2045-2322; doi: 10.1038/srep06340
Other selected publications:
- H. Haick, M.Ambrico, T.Ligonzo and D. Cahen, Controlling Semiconductor/Metal Junction Barriers by Incomplete, Nonideal Molecular Monolayers Journal of the American Chemical Society 128, 6854-6869 (2006) ISSN: 0002-7863; doi: 10.1021/ja058224a
- M. Ambrico, P.F.Ambrico, A.Cardone, T.Ligonzo, S.R.Cicco, R.Di Mundo, V. Augelli, G.M. Farinola, Melanin Layer on Silicon: an Attractive Structure for a Possible Exploitation in Bio-Polymer Based Metal-Insulator-Silicon Devices, Advanced Materials, 23,3332-3336,(2011) ISSN: 0935-9648; doi: 10.1002/adma.201101358
- M. Ambrico, A.Cardone, P.F,Ambrico, T.Ligonzo, V.Augelli, S.R.Cicco, G.M. Farinola, M.Filannino, G. Perna and V.Capozzi. Hysteresis-type current–voltage characteristics in Au/eumelanin/ITO/glass structure: Towards melanin based memory devices, Organic Electronics 11,1809-1814, 2010 ISSN: 1566-1199; doi: 10.1016/j.orgel.2010.08.001
- M. Tulliani, A. Cavalieri, S. Musso, E. Sardellad, F. Geobaldo; Room temperature ammonia sensors based on zinc oxide and functionalizedgraphite and multi-walled carbon nanotubes; Sensors and Actuators B 152 (2011) 144–154 ISSN: 0925-4005; doi: 10.1016/j.snb.2010.11.057
- V. Sreekanth, Antonio De Luca & Giuseppe Strangi Experimental demonstration of surface and bulk plasmon polaritons in hypergratings Scientific Reports (Nature Publishing Group) 3, 03291 (2013) ISSN: 2045-2322; doi: 10.1038/srep03291
- M. Bianco, A. Aloisi, V. Arima, M. Capello, S. Ferri-Borgogno, F. Novelli, S. Leporatti and R. Rinaldi Quartz Crystal Microbalance with Dissipation (QCM-D) as tool to exploit antigen-antibody interactions in pancreatic ductal adenocarcinoma detection Biosensors and Bioelectronics 42, 646–652 (2013) ISSN: 0956-5663; doi: 10.1016/j.bios.2012.10.012
- E. Primiceri, M. S. Chiriacò, E. D’Amone, E. Urso, R. E. Ionescu, A. Rizzello, M. Maffia, R. Cingolani, R. Rinaldi and G. Maruccio, Real-time monitoring of copper ions-induced cytotoxicity by EIS cell chips, Biosens. Bioelectron. 25, 2711-2716 (2010) ISSN: 09565663; doi: 10.1016/j.bios.2010.04.032
- M. S. Chiriacò, E. Primiceri, E. D’Amone, R. E. Ionescu, R. Rinaldi and G. Maruccio, EIS microfluidic chips for flow immunoassay and ultrasensitive cholera toxin detection, Lab on a Chip 11, 658-663 (2011). ISSN: 1473-0197; doi: 10.1039/c0lc00409j
- E. Primiceri, M. S. Chiriacò, F. Dioguardi, A. G. Monteduro, E. D’Amone, R. Rinaldi, G. Giannelli and G. Maruccio, Automatic transwell assay by an EIS cell chip to monitor cell migration, Lab on a Chip 11, 4081-4086 (2011). ISSN: 1473-0197; doi: 10.1039/c1lc20540d
- M. S. Chiriacò, E. Primiceri, A. Montanaro, F. de Feo, L. Leone, R. Rinaldi and G. Maruccio, On-chip screening for prostate cancer: an EIS microfluidic platform for contemporary detection of free and total PSA, Analyst 138, 5404-5410 (2013). ISSN: 0003-2654; doi: 10.1039/c3an00911d