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Hybrid Molecule-Nanocrystal Assemblies for Photonic and Electronic Sensing Applications

Final Report Summary - HYSENS (Hybrid Molecule-Nanocrystal Assemblies for Photonic and Electronic Sensing Applications)

Executive Summary:
1 Publishable summary
1.1 Executive summary
The Hysens consortium has focused on assembly of novel hybrid structures based on inorganic nanocrystals and organic functional molecules for the development of new high knowledge-content hybrid materials (medium term) together with the development of cost effective, scalable processes for controlled production and assembly of such materials at technologically relevant substrates (medium term) with the long term goal of opto-electrical sensing device development. Applications envisaged comprise the water industry and the clinical diagnostic field.
The HYSENS consortium represents a world class interdisciplinary research team with leading experts in the field of synthesis and characterisation of functional nanocrystals and organic components (UNIBAS, UNIBO, BHAM, Tyndall-UCC, TUM) and combined with the expertise in the integration of new functional materials at technologically relevant substrates (UVEG, SCRIBA, CELLIX, MILDENDO). The consortium represents a good balance between industry (three SMEs), research centre (Tyndall-UCC) and fundamental research (five universities).
Research outputs. Excellent progress has been made on all the ambitious project objectives, through focused interactions between partners. Over the 36 months of the project, the collaborative research programme has yielded a very significant number of high quality outputs, including:
• 3 patent applications
• 38 peer reviewed publications published to date with additional publications in preparation
• 5 PhD thesis defended
• 37 oral or poster presentations
Progress overview. Significant progress has been made towards the ambitious scientific objectives of the project. Highlights include:
• Design and successful synthesis of a series of building blocks constituted by inorganic nanocrystals and organic ligands. Organic ligands were engineered to bear anchoring groups for inorganic nanocrystals and functional groups for the selective complexation of group I and II cations, heavy cations and anions. The inorganic nanocrystals were engineered to transduce ion sensing promoted by the organic ligand moiety into an optical or electrical read-out.
• Assembly and characterization of four classes of hybrid structures.
• Demonstration of luminescent sensing in water and serum, showing high affinity for heavy metal ions such as Pb2+ and Cu2+, with limits of detection (LODs) below 10 and 1 g/L, respectively. Sensors based on the modulation of scattering intensity of inorganic nanoparticles were also developed with demonstrated sensitivity for Hg2+ < 200 g/L.
• Fabrication of ion-selective organic electrochemical transistors (IS-OECTs) and demonstration of electrochemical detection of K+ in the mM range.
• Demonstration of electrical detection of Na+ with 100 g/L LOD on Si nanowire FETs. Multiplex cation/anion detection (Na+/F-) in water and serum achieved with Si nanowire FETs integrated into microfluidic flow cells.
• Fabrication of optical sensor based on a miniaturized data matrix code En-TAG™ with built in optical reader for fluorescence and scattering read out. Demonstration of fluorescence detection of Na+ and initial scattering detection of Cu++ with mg/L LODs.
1.2 Project context and objectives
The main objective of the HYSENS project is to exploit organic functional molecules and inorganic nanocrystals as building blocks for the assembly of novel smart materials for detection of Group I, II, or transition metal cations and anions in water and artificial serum matrices. The motivation behind the choice of those matrices is the following: the increasing constraints on water supply has led to the implementation of recycling plants for both potable water and technological applications, increasing the demand for low cost and rapid contaminant detection (and elimination) technologies. This presents a significant problem for existing and future water-dependent industries which require both expensive ultrapure water and water analysis systems. The technology developed by HYSENS is targetted towards meeting these new demands for cheap and efficient analysis of water contaminants. Practical technological applications will be 1) the semiconductor industry where routinely inorganic cations in ultrapure water need to be detected at concentrations down to (and below) the ng/L range, presently using highly expensive inductively coupled plasma mass spectrometry (ICP-MS); 2) clinical diagnostic areas where there is an increasing demand for the development of innovative low cost electrolyte analysis technologies that could be applied, for example, in emergency rooms to obtain fast indication for the diagnosis of specific diseases. In this field, existing techniques meet concentration specifications required. However, more accurate, selective and sensitive methods would revolutionise the field of diagnostics for early detection and management of renal, endocrine, acid-base, water balance disorders, and many other conditions.
1.2.1 Objectives
The proposed innovation in HYSENS relies on the use of hybrid inorganic-organic materials. The hybrid material intelligence resulting from the engineered combination of individual components will allow the coherent execution of functionalities allowing us to reduce false sensing outputs, leading to the development of sensors with enhanced selectivity and sensitivity. Inorganic nanocrystals and organic functional molecules will be used for the assembly of four novel classes of hybrid nanostructures: intelligent hybrid assemblies 1, PRET hybrid assemblies 2, 2D hybrid arrays 3, and 1D hybrid arrays 4. The proposed hybrid structures have been chemically programmed to function as sensors for group I, II and transition metal cations (Na+, Ca2+, Cu2+) and anions (F-, PO43-). The four main objectives of the project are summarised below:
• Semiconductor nanocrystal - organic molecule “intelligent hybrid assemblies” (intelligent assembly 1) for demonstration of optical “intelligent chemosensing” (objective 1) targeting ng/L detection of cations and anions (including Na+, Ca2+, Cu2+, F-, PO43-).
Industrial validation of intelligent assembly 1 into polymer-patterned tag surfaces and microfluidic cells.
• Metal nanocrystal – organic molecules “PRET hybrid assembly” (intelligent assembly 2) for demonstration of optical sensing based on plasmon resonance energy transfer (PRET) mechanisms (objective 2) targeting ng/L detection of transition metal ions, including Hg2+ and Cu2+. Industrial validation of PRET intelligent assembly 2 into polymer microfluidic cells.
• Metal nanocrystals - organic molecules “2D hybrid array” (intelligent assembly 3) for demonstrating large area sensing (objective 3) with an emphasis on targeting electrical “intelligent chemosensing” of Group I and II cations and anions (including Na+, Ca2+, F-, PO43-). with ng/L detection limits.
• Metallic, semiconductor nanocrystals - organic molecules (objective 3) interfaced on 1D Si FET arrays, “intelligent assembly 4”, for electrical readout based sensing (objective 4) and electrical “intelligent chemosensing” of Group I and II cations and anions (Na+, Ca2+, F-, PO43-) at ng/L levels. Industrial validation involving integration of polymer microfluidic cells in the 1D hybrid arrays 4.
1.2.2 Work Breakdown Structure
WP1: Synthesis and characterisation of inorganic and organic building blocks
WP2: Assembly and characterisation of hybrid organic functional molecule-nanocrystal structures
WP3: Optical sensing functionality in hybrid structures 1, 2
WP4: Electrical sensing functionality in hybrid structures 2, 3, 4
WP5: Industrial Validation
This is an overarching technical WP focusing on industrial validation of processes and methodologies from WPs 1- 4 targeting development methodologies for the interfacing of hybrid assemblies on:
(i) patterned polymer test structures (objective 1)
(ii) electrochemical cells for electroluminescent readout sensing (objective 1)
(iii) plastic microfluidic test structures (objectives 1, 2, 4)


Project Context and Objectives:
1.3 Work progress and achievements during the period
1.3.1 WP1: Synthesis and Characterisation of Building Blocks.
WP leader: UNIBAS-Chem.
Overview. Work package 1 is the start of the materials pipeline for HYSENS and is tasked with the delivery of new materials for the detection of the selected analytes. The primary tasks of the work package are
• the design of materials for the detection of the identified analytes
• the further molecular engineering of functionalisation of these materials to optimise stability for the selected working environment and to incorporate binding motifs appropriate to the selected carrier modalities (surfaces, nanoparticles etc.)
• synthesis of the newly designed materials
• synthesis of optimised nanoparticles
• preliminary evaluation of the properties and binding capacity of the new materials
• evaluation of scale-up feasibility
• delivery of materials to subsequent work packages in the pipeline.

Deliverables outstanding: none
WP1 conclusions. The molecular targets were designed on a modular basis and incorporated three primary modules: (i) the molecular recognition module optimised for the binding of the ion of interest with a concomitant readable signal (ii) an anchoring module for binding to the surface of nanoparticles or other surfaces and (iii) a linker connecting modules (i) and (ii) that might be innocent or optimised for electronic or energy transfer between the two. Synthetic highlights include the design and successful preparation and delivery of compounds with high selectivity for sodium, calcium and anions. All of these materials were also optimised for binding to surfaces and/or nanoparticles. In the course of the work, new synthetic methodologies had to be developed, in particular an in situ deprotection method that was optimised and patented. Novel methodologies for the formation of metal nanorod ordered assemblies showing enhanced optical properties were developed and patented. In the course of the work also novel methodologies for the self-assembly of anisotropic metal nanoparticles were developed and patented.
WP1 Summary of achievements
Task 1.1 Nanocrystal synthesis
The synthesis of luminescent semiconductor nanocrystals within this task has been carried out by UNIBO. In the first part of the project the efforts were focused on the preparation of ZnTe and ZnTe-ZnS core-shell nanocrystals. Techniques such as energy-dispersive X-ray (EDX) and high resolution TEM investigations have been employed in combination with XPS and UV-vis spectroscopies for the assessment of the composition and morphology of the prepared materials. ZnTe Nanocrystals of 7-10 nm diameter have been obtained with good size control but their stability is poor and they show no luminescence. A route for the controlled growth of a ZnS shell on the ZnTe core has been identified, yielding the first ZnTe-ZnS core-shell quantum dots ever made. As expected, the ZnS shell improves the chemical stability of the nanocrystals, which however remain not emissive and rather sensitive to the environment. These particles are more environmentally friendly than common cadmium-based quantum dots but are not suitable for incorporation in the intelligent hybrids 1 because they are not luminescent. Nevertheless, they are interesting nanomaterials because of their properties and the non-trivial chemistry associated with their size-controlled preparation.
The activities of UNIBO in this Task have thus been concentrated on the production of high quality, strongly luminescent CdSe-ZnS core-shell nanocrystals to be used for the assembly of intelligent hybrids 1 (WP2), for the construction of LECs (WP3) and as reference/model components for sensing experiments (WP3), or made available to other consortium partners. Two methods for the upscalable production of core-shell nanocrystals with a satisfying size selectivity and monodispersion have been optimized.
Method A: SILAR. For larger quantum dots (dcore > ca. 4 nm), the classical successive ion layer adsorption and reaction (SILAR) method, using zinc oxide and elemental S as precursors, has been successfully employed to deposit a precisely controllable number of ZnS shell layers onto CdSe cores. Hydrophobic nanocrystals with intense emission in the orange-red spectral region can be prepared.
Method B: one-pot. For smaller nanocrystals (dcore < ca. 4 nm), the use of the SILAR method results either in the degradation of the particles during the preparation of the reactive mixture or in a significant red shift of both the absorption and the emission spectra of the quantum dots during the deposition of the first monolayer. To avoid these undesirable effects, a one-pot method consisting of the deposition of the entire shell at once has been adopted. Diethylzinc (ZnEt2) and bis-trimethylsilylthiane (TMS) have been used as the sources of zinc and sulfur, respectively. These precursors are more reactive than oleic acid-activated ZnO and elemental sulfur used in the SILAR approach, thereby allowing a significant decrease of the coating temperature and limiting side reactions. CdSe-ZnS nanoparticles emitting in the green-yellow region have been synthesized by this approach.
In summary, these methodologies have enabled UNIBO to synthesize and isolate high quality, strongly luminescent CdSe-ZnS (2-5 shells) quantum dots with size varying from 3 to 10 nm and an emission band finely tunable from 525 nm (green) to 700 nm (red). Because of these properties and their excellent chemical and photochemical stability, such nanocrystals are very suitable for the production of intelligent hybrids 1.
Task 1.2. Synthesis of organic functional molecule building blocks
Initially, HYSENS identified a range of "simple" ionic analytes as targets for detection in aqueous, point-of-care, environments, including sodium, potassium, calcium, phosphate, nitrate and (possibly) fluoride ions. Tasked with these demands, WP1 designed a series of prototype molecular systems containing molecular recognition motifs appropriate to these targets and anchoring groups designed for metal chalcogenide nanaoparticles or noble metal surfaces (Figure 1).

Figure 1. Initially identified target molecular materials for the detection of group 1 metal ions (C1, C3), group 2 metal ions (C2), heavy metal ions (C4, C5) and anions (C6,C7,C8).
According to plan, the targets were refined according to likely impact for point of care tests, ease of synthesis, up-scalability and the intellectual property environment at M3. UNIBAS-CHEM was tasked with the synthesis of C1, C2 and C8 and BHAM with C4 and C5. Minor structural changes to the target molecules were made to facilitate synthesis without changing the overall concept and the target analytes.
C1 has been prepared in gram and multigram quantities by UNIBAS-Chem in a very simple process that is optimised for upscaling at an overall 35-40% yield (Figure 2).
C1 was identified as one of the lead compounds for carrying through the materials pipeline and identifying the end-use market as well as preparing demonstrator devices with point-of-care sodium quantification.

Figure 2 Synthesis of C1, a sodium detector using a simple amide coupling of the molecular recognition motif ( a crown ether) to the anchor through an amide coupling reaction with lipoic acid.
C2 has been prepared in multigram gram quantities and upscaling has been optimised. .
BHAM were given the objective of synthesising the organic functional molecules; C4 and C5 for the purpose of sensoring transition metal ions; Cu2+ and Hg2+. Both molecules were synthesized and distributed to partners.
The target compound C8 was structurally modified for synthetic ease and has been prepared in gram quantities and delivered to partners (Figure 3).

Figure 3. Synthesis of the ruthenium complex designed for detection of fluoride (and other) anions.
Next generation molecular components
A new family of ligands (Figure 4) has been developed in which a 2,2':6',2''-terpyridine metal-binding domain is conjugated with an anchoring group appended through a short but flexible chain. This family was selected on the basis of synthetic accessibility and electronic isolation of the anchor from the metal-binding domain. The long-term consideration was performing FRET experiments on surfaces. The top row of the figure illustrates the compounds in their as-synthesised protected forms which can be stored indefinitely and delivered without special precautions to partners and end-users. The bottom row shows the deprotected versions which are highly specific for particular surface types but which suffer from the associated high reactivity in terms of long-term stability.

Figure 4. Next generation tpy ligands for energy transfer applications.


1.3.2 WP2: Assembly and characterisation of hybrid organic functional molecule-nanocrystal structures
Leader: UBham
Overview
Work package 2 comprises the assembly of inorganic nanocrystals and organic functional molecules synthesized in WP 1, into four novel classes of hybrid nanostructures: intelligent assembly 1, PRET hybrid assemblies 2, 2D hybrid arrays 3 and 1D hybrid arrays 4. The main tasks of the work package are the assembly of such structures and initial evaluation of their formation and stability through optical and electrical characterization.

Deliverables outstanding: none
WP2 conclusions. Assembly of the following hybrid structures was demonstrated: intelligent assembly 1 (C1 and C2), PRET assembly 2 (C5). The formation of the assemblies was demonstrated by optical microscopy. Fabrication of Si nanowire FETs is complete and assembly of 1 hybrid array 4 (C1, C8*) is complete. All assembled hybrids have been supplied to industrial partners for industrial validation.
WP2 achievement summary
Task 2.1. Assembly of and Characterisation of Intelligent Hybrids 1 1 (objective 1)
Owner UNIBO:
This task deals with the assembly of inorganic nanocrystals and organic functional molecules synthesized in WP1 and the characterization of the resulting hybrids. The general strategy for the assembly involves the replacement of the alkylamine/alkylphosphine monolayer covering the surface of as-synthesized nanocrystals with a layer of the desired functional ligands. UNIBO, in collaboration with UNIBAS-CHEM, has developed a handy and efficient methodology (patent pending) that enables the exchange of the native hydrophobic ligands of semiconductor nanocrystals with ligands containing the 1,2-dithiolane unit as the surface docking group, such as those developed in Task 1.2. This procedure does not make use of metal ion salts and is therefore suitable for the preparation of chemosensors for metal ions. The method is based on an anionic Amberlite resin loaded with borohydride as the reducing agent to transform 1,2-dithiolane into bis-thiol, which successively binds to the nanocrystal surface. The occurrence of the cap exchange reaction is signalled by the phase transfer of the nanoparticles from the apolar organic solvent to methanol or water. Finally, the nanocrystals suspension is filtered with a syringe filter (0.46 m pore size) to remove possible large aggregates, and successively purified with 3 cycles of dilution/concentration with a centrifugal filter (Amicon Ultra-0.5 mL, 30 kDa, 7000 rpm, 12 minutes) to eliminate the excess of free ligand. The hybrids have been characterized by TEM and absorption and luminescence spectroscopy, confirming that the nanocrystals are intact and that the functional ligands are attached to the nanocrystal. The average number of functional ligands (C1, C2) per nanoparticle could has been estimated from absorption data.
UNIBO has used this methodology to carry out the exchange of the hydrophobic ligands of CdSe-ZnS nanocrystals with the C1, C2, TA-PEG400 and TA-TEG ligands, all containing the 1,2-dithiolane unit. As C2 is provided as a tetra-ester, the deprotection of the four carboxylic acid moieties of the receptor has to be performed prior to QD surface functionalization. The optimized deprotection reaction affords pure C2 in its tetracarboxylate form (that is, C24–) in 70-95% yield; once C24– is isolated, the functionalization of the QDs can be performed using the usual protocol. However, due to the presence of the four carboxylate groups, C24– interacts with the borohydride resin after reaction with BH4–, and the use of an ionic salt is needed to extract the reduced bis-thiol ligand from the resin.
Several batches of intelligent hybrids 1 have been produced and forwarded to WP3 for sensing studies or have been made available to the consortium. Hybrids containing from 10 to 1500 C1 or from 10 to 100 C2 molecules per nanocrystal were produced using CdSe-ZnS core-shell nanocrystals of different size (color), although the best results were obtained with yellow-orange emitting particles. Nanocrystals coated with unsubstituted lipoic acid have also been prepared for reference purposes.
Intelligent hybrids functionalized with a mixed layer of C1 and C2 functional ligands (in addition to PEG ligands to afford water solubility) have been prepared with the aim of implementing combined Na+/Ca2+ sensing with logic response. Samples of “ternary” hybrids QD-C1/C2/PEG have been obtained with an approach similar to that employed for functionalization with either C1 or C2. In all cases the hybrids are water soluble and highly luminescent. UV-vis spectroscopic experiments indicate the presence of rougly equal amounts of C1 and C2 ligands on the surface of the particles, ranging from 10 to 100 molecules per nanocrystal.
Task 2.2 Assembly and Characterisation of PRET Hybrids (Objective 2)
Owner Tyndall-UCC:
Three types of PRET hybrids have been assembled and characterised by Tyndall: C5 functionalised Au nanoparticles (NP), C5 functionalised pseudo Ag core–Au shell nanoparticles and C8* functionalised Au NP. The first hybrid is the original proposed hybrid, the second was designed to improve the overlap between NP scattering and C5 absorption bands, the third was chosen for the good overlap between Au NP scattering and C8 absorption bands. Steps of the assembly were: silanisation of glass substrates, immobilisation of NP on the substrates and functionalisation of the NP. The silanisation of the glass substrates has been characterised by ellipsometry in collaboration with BHAM confirming a monolayer formation of APTES (3-aminopropyltrimethoxysilane). Subsequent immobilisation of NP on APTES with suitable NP density was confirmed by optical dark field microscopy. The pseudo core-shell NP were fabricated by Au evaporation onto immobilised Ag NP. The functionalisation of NP was confirmed by indirect methods, mainly dark field spectroscopy, where a red shift of the NP scattering band confirmed the attachment of molecules on the surface of the NP (10-15 nm shift for C5, 5-10nm shift for C8*). Additionally fluorescence and Raman spectroscopy were used to support the dark field spectroscopy findings where both methods showed characteristic peaks for the molecule attached.
Task 2.3 Assembly and Characterisation of 2D Hybrid Arrays 3 1 (objective 3)
Owner UNIBAS-PHYS:
Au nanoparticles (10nm core diameter) were synthesized by UNIBAS-Phys. Using a PDMS stamping technique, well-ordered nanoparticles arrays (2D hybrid arrays 3) were successfully prepared and studied. Organic functional molecules (C1) delivered by partner UNIBAS-Chem were attached on the nanoparticle array and characterized by electrical means. As a major result, UNIBAS-Phys demonstrates succesful insertion of the C1 molecules into the arrays.
Task 2.4 Fabrication of Si nanowire FETS (objective 2)
Owner UNIBAS-PHYS:
Within the HYSENS project, UNIBAS-Phys investigated different approaches for the detection of ions in water based solutions using silicon nanowires field-effect transistors (Si NW FETs). The goal within work package 2 was first to achieve high-quality FETs and demonstrate sensing functionality.
In collaboration with PSI Villigen, low leakage, low noise and small hysteresis FETs with Al2O3 and HfO2 as gate dielectrics were successfully fabricated using a top-down fabrication process on silicon on insulator (SOI) wafers. Figure 1 (a) – (c) shows optical pictures of a final device. Each sample consists of 48 nanowires.
Furthermore, the sensing functionality of Si NW FETs was demonstrated. Responses at the theoretical limit were achieved in case of pH.
Task 2.5 Assembly and Characterisation of 1D Hybrid Array 4 (objective 2)
Owner UNIBAS-PHYS:
To achieve a platform for the functional molecules developed by the HYSENS consortium, the wires were successfully covered by either a thin gold film or gold nanoparticles. Figure 1 (d) – (f) shows SEM graphs for a nanowire with oxide surface (d), a nanowire coated with a thin gold film (e) and a nanowire which is decorated with gold nanoparticles (f).
As a result, a high-quality and flexible platform was achieved for specific ion detection which is further investigated in work package 4.

Figure 5 (a) Optical microscope image of a chip containing four arrays with twelve SiNW FETs each. Inset: Photography of the final sensor device with 48 nanowires of different size. (b) Zoom to one half of a nanowire array showing six nanowires sharing one drain contact. (c) 3 nanowires of 100nm width. The liquid channel crosses the wires vertically. (d) Electron beam micrograph of a nanowire with 100nm width and Al2O3 as top surface. The dark regions at the contacts show the ion implantation. (e) Electron beam micrograph of a gold coated nanowire. (f) Electron beam micrograph of a nanowire (bright region) decorated by gold nanoparticles.

1.3.3 WP3: Optical sensing functionality in hybrid structures 1, 2. Leader: UNIBO
Overview
The aim of workpackage 3 is the initial assessment of sensing functionality in intelligent assembly 1 and PRET assembly 2. Prototype intelligent assemblies 1 are expecting to display sensing functions with optical (fluorescence quenching upon analuyte binding) readout due to interactions occurring between the nanocrystal unit and the organic multicomponent unit, regulated by analyte binding.. A parallel study lead by UVEG is devoted to the incorporation of intelligent assemblies 1 into light emitting electrochemical cells (LECs) that can change their electroluminescence properties (intensity and/or colour) upon analyte detection. The readout for PRET assemblies 2 is based on modification of nanocrystal Rayleigh scattering light emission, regulated by analyte binding. Main tasks for this workpackage are:
• Investigation of the optical response of Na+ ions on intelligent assemblies 1
• Fabrication of LECs
• Incorporation of intelligent assemblies 1 or prototype components into LECs and initial characterisation of devices
• Investigation of the optical response of Hg2+ ions on PRET assemblies 2
• Initial incorporation of intelligent assemblies 1 into optical tags

Deliverables outstanding: none
WP3 progress update. Investigation of the sensing performances of intelligent assembly 1 with C1 and C2 were investigated for sensing towards group I and II metal cations. Also the sensitivity towards heavy metal was explored with good LODs found in water as well as in an artificial serum matrix. Logic sensing for mixed-ligand assembly (C1+C2) towards Na+ and Ca2+ was also investigated.
Several prototype microfluidic structures were tested in order to be assembled with the optical sensing element (LEC). A microfluidic structure provided by Cellix has been successfully integrated with working LECs by lamination, and the optical and electrical response of the device to aqueous and non-aqueous ionic solution was measured. In order to pursue ion sensitivity and low detection limit, UVEG decoupled the optical from the electrochemical response, and fabricated electrochemical sensors with low detection limit and high selectivity to a specific target ion. Very high sensitivity was achieved by using conducting polymers in contact with a gel electrolyte and integrated with state-of-the-art ion-selective membranes.
PRET assembly 2 with C5 and C8* was tested for detection of Hg(II) and F-. Initial detection in the range of mg/L was obtained. Investigation of Hg(II) detection by amalgamation processes has been investigated. Fast (10 min) and low LOD (< 200 g/L) in water solutions have been achieved. Sensitivity and selectivity are been investigated.
WP3 conclusions. Intelligent hybrids 1 with C1/PEG or only PEG ligands were found to behave as luminescent sensors for a variety of group I and group II metal cations, although with a poor selectivity (D3.1). Intelligent hybrids 1 with C1/PEG or only PEG ligands were found to exhibit a very high affinity for heavy metal ions such as Pb2+ and Cu2+, with limits of detection (LODs) below 10 and 1 g/L, respectively (D3.1). Although the luminescence quenching of the nanocrystals in response to the metal ion concentration is irreversible, such nanohybrids can be pratically employed to evidence the presence of Pb2+ and Cu2+ impurities in water down to trace amounts. Intelligent hybrids 1 with C2/PEG showed a remarkable sensitivity for Ca2+, with LODs in the g/L, range (D3.1). The selectivity of the Ca2+ receptor embedded in the C2 is retained when this ligand is bound to the QD surface. As a result, the QD-C2/PEG400 hybrids exhibit a marked selectivity for Ca2+ with respect to K+ and Mg2+, two of its principal metal ion competitors.
PRET assembly 2 with C5 and C8* was tested for detection of Hg(II) and F- (D3.5). Initial detection in the range of mg/L was obtained (D3.6). Fast (10 min) and high sensitivity (< 200 g/L) detection of Hg(II) in water solutions have been achieved by amalgamation processes.
Novel electrochemical cells based on cyanine dyes and electrochemical sensors integrated into microfluidic cells have been fabricated. The latter has shown low detection limit and high selectivity towards model ion K(I) (D3.3).
WP3 summary of achievements
Task 3.1. Fluorescence sensing performance assessment of intelligent assemblies 1 (objective 1)
Intelligent hybrids 1 with C1/PEG or only PEG ligands were found to behave as luminescent sensors for a variety of group I and group II metal cations, although with a poor selectivity. The luminescence response of hybrid 1 towards Na+ was validated on polymer tag surfaces using a dedicated optical reader.
The same hybrids were found to exhibit a very high affinity for heavy metal ions such as Ni2+, Hg2+, Pb2+ and Cu2+, with limits of detection (LODs) below 50 g/L (2 g/L for Cu2+). Although the luminescence quenching of the nanocrystals in response to the metal ion concentration is irreversible, such nanohybrids can be practically employed to evidence traces of heavy metal ions impurities in water.
Intelligent hybrids 1 with C2/PEG showed a remarkable sensitivity for Ca2+, with LODs in the sub-g/L, range. The selectivity of the Ca2+ receptor embedded in the C2 is retained when this ligand is bound to the QD surface. As a result, the QD-C2/PEG hybrids exhibit a marked selectivity for Ca2+ with respect to K+ and Mg2+, two of its principal metal ion competitors (Fig. BO1).


Figure BO1. (a) Luminescence spectral changes and (b) titration curve observed for the hybrids QD-C2/PEG400 (110–8 M) upon addition of Mn+Cl–n (Mn+= Ca2+, K+, Mg2+) in water.
The mixed-ligand intelligent assembly QD-C1/C2/PEG showed a combined response towards Ca2+ and Na+. Such a behavior highlights the capability of these hybrids to determine two different analyte targets according to a logic function.
The QD-PEG hybrids exhibit a highly sensitive luminescence response towards Fe2+ and Hg2+ (selected as representative examples of physiologically relevant and harmful ions, respectively) in an artificial serum matrix. The LODs for Fe2+ and Hg2+ in serum are as low as 7 nM (0.4 g/L) and 10 nM (2.4 g/L); considering that the serum iron cutoff level is 15 g/L and the normal mercury blood level is <5-20 g/L, these performances are of interest for blood tests.
Task 3.2 Fabrication of electrochemical cells (LECs) (Objective 1)
Intelligent assembly 1 was integrated in light emitting electrochemical cells (LECs). Specifically, charge transporting matrices based on ionic transition metal complexes (iTMCs) were used to in combination with QDs as the active layer of the electroluminescent device. All possible configurations were explored, i) mixed layer of QDs and iTMCs, ii) pure QDs layer and iii) pure iTMC layer. While no energy transfer from the iTMC to the QDs was observed in the mixed layer configuration (neither by photo- nor electroluminescence), electroluminescence from solution processed QDs and iTMC pure layers were recorded. Although the iTMCs based on IrIII supplied by UNIBAS-CHEM showed high photoluminescence quantum yield (PLQY = 19%) and lead to bright LECs (>100 cd/m2), other charge transporting matrices and light-emitting materials were explored. For the first time, high PLQY was obtained for host–guest films using two cyanine dyes, reaching 27%. Due to the ionic nature of the dyes, the devices operate according to the mechanism established for LECs, allowing the use of air-stable electrodes and very low operation voltages. Sandwiching these films in between two electrodes allows for very stable near-infrared emission with a maximum radiant flux of 1.7 W m−2 at an external quantum efficiency of 0.44%. As cyanine dyes are easily adjusted and prepared in large quantities, they constitute an interesting class of ionic emitting materials to be employed in solution-processed optoelectronic devices.
Task 3.3 Optical sensing with electroluminescence readout of intelligent assemblies 1 (objective 1)
LECs were fabricated with structured top-electrodes (finger electrodes), in order to allow liquids to access the active layer in the light-emitting zone of the devices. Microfluidics structures provided by partners (SCRIBA and CELLIX) were successfully integrated with LECs into optical sensing platforms.
For the monitoring of the sensor parameters, a custom made system was built, consisting in a device holder with the electrical connections to the sensors and equipped with: i) a photodiode to quantify the electroluminescence; ii) a video camera able to record in real time the local electroluminescent cell response; iii) microfluidic inlet and outlet controlling flux of analyte into the sensor. The sensor was evaluated with diluted K+ and Na+ solutions, using both water and ethanol as solvents, showing limited sensitivity and selectivity.
In order to enhance the device performance, the optical and electrochemical sensing mechanisms were decoupled and a purely electrochemical sensor was prepared. The device, an organic electrochemical transistor (OECT), makes use of conducting polymers as the active layers and can be used in direct contact with water without substantial degradation. By using an ion-selective membrane made of polyvinylchloride doped with a potassium ionophore, low detection limit (10–6 M), high selectivity and sensitivity (> 50 A/dec) for potassium in aqueous solutions was demonstrated. These values exceed what previously reported for ion-selective transistors, and can be immediately applied to human diagnosis as well as environmental and water monitoring.
Task 3.4 Optical sensing performance assessment of PRET assemblies 2 (objective 2)
Two types of PRET assemblies have been assessed in terms of optical sensing. Pseudo Ag core – Au shell nanoparticles (NP) functionalised with C5 for Hg2+ detection in EtOH (PRET 2.1) and Au NP functionalised with C8 for F– detection in MeCN (PRET 2.2). For reasons of optical stability of the sensing platform throughout the measurement all measurements have been carried out in microfluidic cells provided by partner Mildendo relating to Task 5.5. For assembly PRET 2.1 we detected an optical response in the form of reduced NP scattering for a Hg2+ concentration of 20 mg/L. However, due to incompatibility of EtOH with the microfluidic cells resulting in cell leakage and floating dirt these results could not be reproduced. Detection of fluoride ions using PRET 2.2 was not successful, caused by insufficient NP functionalization or failed plasmon resonance energy transfer.
On an alternative route we demonstrated Hg2+ detection using non-functionalised Au nanorods (NR) based on amalgamation of the NR that leads to a colour shift of the NR scattering. With single NR spectroscopy we were able to detect Hg2+ concentrations down to 0 g/L after immersion in the analyte solution for 10 min. For facilitated read-out first test with dense NR substrates that can be read in simple transmission or reflection and with laser patterned Au NR substrates designed for read out with a reader developed by Scriba have been carried out. For both substrates the detection of 200 g/L Hg2+ could be achieved with optical spectroscopy.

1.3.4 WP 4: Electrical Sensing Functionality in Hybrid Structures. Leader: TUM
Overview
WP 4 is the electrical and optoelectrical characterisation of PRET assembly 2, 2D assembly 3 and hybrid array 4. Analogously to WP3, the above hybrid structures are expected to display and electrical readout that change upon analyte binding. The main tasks for the workpackage are:
• Assembly of metal nanostructures (PRET 2) into superstructures for photoconductivity measurements
• Photoconductivity measurements on PRET 2
• Electrical measurements on 2D array3
• Electrical functionality on Si NW FETs

Deliverables outstanding: none
WP4 conclusions. Assembly of 2D hybrid arrays 3 completed and demonstration of molecular photoconductance (D4.2). Electrical sensing functionality of Silicon nanowire FETs (D4.3) investigated by studying pH response of Si nanowires with different gate dielectrics. Demonstration of the selective detection of Na(I) using 1D hybrid array 4 (C1) molecules anchored on a gold film interface (D4.4). Similarly, C2 and C8* molecules were also successfully utilized for the selective detection of Ca2+ and F- ions upon incorporation of 1D hybrid array 4 into microfluidic cells (D4.6) (100 g/L LOD).
WP4 summary of achievements
UNIBAS-PHYS has developed know-how on the fabrication and fundamental working principles of silicon nanowire field-effect transistors (SiNW FETs). In the past years, these devices have been demonstrated to be good pH sensors. However, their use for specific ion sensing other than protons remains a challenging task requiring novel interfaces and surface treatments. The goal of work package 4 was the specific detection of selected ions such as sodium Na+, potassium K+, calcium Ca2+, fluorine F- and copper Cu2+ in water. To achieve this goal, UNIBAS-Phys functionalized nanowires either directly on the oxide surface, via an additional thin gold film or using polymer membranes. The gold film provides an alternative surface for anchoring molecules developed within the HYSENS consortium. These molecules act as receptors which specifically trap the targeted ion. In another approach, the gate dielectrics of Si nanowires FETs was decorated with gold nanoparticles. Using nanoparticles instead of a thin gold film expands the possibility of electrical readout to optical methods investigated by partner TUM.
As major achievements, UNIBAS-Phys, together with partner UNIBAS-Chem, demonstrated the selective detection of sodium ions using designed molecules anchored on a gold film interface. Also K+, Ca2+ and F- could be detected successfully. Within the HYSENS project, UNIBAS-Phys has developed a versatile sensing platform and gained deep insight into the fundamental processes present in the system. As a result, the proposed approach based on SiNW FETs was successfully used as a sensing platform and simultaneous detection of different targeted ions has been demonstrated.
TUM has developed a fundamental understanding of the optoelectronic read-out of test structures with metal nanoparticles and molecules incorporated. The revealed mechanisms comprise bolometric, molecular, and plasmonic excitations considering the morphology and the charging energy of the nanoparticle structures. The molecules were provided by UNIBAS-CHEM/PHYS and BHAM. The measurements were partly accompanied by an electrical characterization of UNIBAS-Phys. Partner TUM developed a FIB lithography to taylor the lateral dimensions of nanoparticle arrays such that the overall optoelectronic response is enhanced. In cooperation with Tyndall-UCC, TUM characterized Au nanopartical structures which were assembled by dielectrophoretic trapping to verify that the optoelectronic signal can be used to read out the plasmonic excitations polarization-sensitive.


Figure 6. Differential measurement setup for sodium (Na+) and potassium (K+) detection. Measuring the difference between a nanowire with a gold surface functionalized by the molecules (active nanowire) and a nanowire with a bare gold surface (control nanowires) helps to minimize background interference coming from additional unspecific surface reactions.




1.3.5 WP5: Industrial Validation. Leader: Cellix Limited
Overview
Workpackage 5 is related to the incorporation of hybrid materials synthesised and characterised in WPs 3 and 4 into test structures towards extension of sensing performances into industrially relevant substrates. Therefore this workpackage comprises the fabrication and customisation of the following test structures: i) polymer optical tags, ii) plastic microfluidic cells. Although no deliverables within WP5 fall in the M1-M18 period, industrial partners have already fabricated first generation of test structures. Preliminary work is being carried out on the incorporation of hybrid materials or hybrid components into these structures with the aim of having optimised test structures ready for incorporation of optimised hybrid materials in the second part of the project. WP5 most relevant results are presented below.

Deliverables outstanding: none. D5.3 has not been submitted as it is due at M36.
WP5 conclusions. The efforts of the industrial partners have been focused on demonstration of optical and electrical sensing of hybrid molecules incorporated into optical tags and microfluidic flow cells. Scriba has demonstrated fluorescence read-out of intelligent assembly 1 in presence of Na(I) with target read out of 200 g/L. patterned metal nanoparticle tags have also been fabricated by Scriba and tested for detection of Cu(II) with PRET assembly 2 (200 g/L read). A novel cell flow was fabricated by Cellix and delivered to UVEG for integration of LECs towards detection of K(I). Electrical sensing of 1D hybrid array 4 into PDMS flow cell was demonstrated.
The first flow cell, designed by Mildendo and Cellix and finally manufactured by Mildendo, has been largely used in Tyndall and Cellix during the optical validation of PRET assembly as this enabled an easier integration of the functionalized glass substrates within the microfluidic structure. The material and dimensions of this flow cell have been defined in order to allow the optical readout via dark field spectroscopy or fluorescent detection. PRET assembly 2 (C1 and C8*) has been incorporated in the flow cell and preliminary detection of Hg(II) and F- has been demonstrated (D5.5).
Cellix has also designed, manufactured and delivered to University of Valencia a second flow cell device which facilitated the optical detection of intelligent assembly 1 with electroluminescent detection. The experiments in UVEG showed promising results using ion-selective organic electrochemical transistors (IS-OECT).
An additional flow cell has been manufactured by UNIBAS Phys for the incorporation of 1D hybrid assembly 4 into microfluidic cells. Detection of Na(I) and F- has been demonstrated (D5.6).
WP5 summary of achievements
The constantly growing market demand for novel and affordable sensing technologies has continuously encouraged the interest of the HYSENS consortium towards the study and development of hybrid materials to be used in a new generation of sensors with improved sensitivity and selectivity. Many routine and quality control applications performed daily in the pharmaceutical, semiconductor, food and water supply industries will enormously benefit from further accurate information of the analysis and will make various processes at research and production levels more effective. The technological efforts made under this grant, have primarily shown the feasibility of several types of miniaturized sensors along with the possibility of integration within microfluidic devices, with the main benefits being accuracy, portability, low sample requirement and low reagent consumption. Considerable progress has been achieved in all aspects of interest extending from the micro-fabrication techniques to innovative chemical processes.
The focus of WP 5 has been the industrial validation of selected sensing structures, identified in a preceding phase of the project, integration of these structures into microfluidic devices and testing to evaluate their functionality.
Scriba has also developed two prototypes of Optical Readers which are capable of decoding the information content of the miniaturized En-TAG™ and subsequently extract the response of the sensor, by looking at the optical contrast of the functional materials.
The analysis of the water matrices related to content of dissolved ions is definitely an application field that gathers considerable interest. Scriba has developed both a low-cost sensor (in the form of a flexible label) with sustainable fabrication process and a tool for decoding of the sensor, based on the properties of innovative photonic materials, integrated additively as a micrometric pattern.
During the course of the project, Scriba has tested two different architectures of the sensor containing either one of the following:
• Fluorescent Quantum Dots functionalized with organic molecules capable to capture sodium and potassium ions in solution (Task 5.1)
• Gold Nanoparticles with organic shells sensitive to the content of copper and mercury ions in water matrices (Task 5.2)
The sensitivity of these sensors has been tested in water environments, with two levels of ionic contamination (Na+ and Cu2+). The analysis have been performed on drinking water samples, and secondly in special ultrapure water normally used during clean room manufacturing processes. The tests were positive, considering the capability of the sensor to detect the presence of dissolved chemical species. The selectivity of the sensor with respect to the type of ions is a peculiar characteristic of the material developed in HYSENS.
The architecture proposed by Scriba also allows you to integrate several sensitive areas on the same miniaturized code En-TAG™, in order to make a parallel read-out and get totally unambiguous response. This will be one of the objectives of the exploitation of the results of HYSENS project, allowing Scriba to propose a low-cost sensor to the market for the detection of heavy metal ions in solution.

Figure 7 a) Design of the detection mechanism, for the integrated sensor En-TAG™ with fluorescent ring of functionalized Quantum Dots. b) Screenshots of change of fluorescence intensity of a sensor exposed to NaCl water solution (10-6 M). c) Snapshot of the Optical Reader (for Quantum Dots) developed by Scriba during the Hysens project.
The workpackage leader Cellix has demonstrated a strong competence in the design and manufacture of microfluidic devices, and acquired further experience in this area by establishing strong cooperation link with all the partners of the HYSENS project. Cellix’s expertise, which extends from the development of precision pumping solutions to cell-based assays and disposable devices that mimic the human vasculature system, benefitted the consortium by producing custom flow cell devices and adapting their existing fluidic platform for the execution of HYSENS experiments. The prototype flow cell, designed by Mildendo and Cellix and manufactured by Mildendo, has been extensively used in Tyndall and Cellix during the optical validation of PRET assembly. The material and dimensions of this flow cell allowed the optical readout via dark field spectroscopy or fluorescent detection. The advancements achieved under the HYSENS grant in the design and micro-fabrication techniques of microfluidic devices are considerable: the prototype biochip design allows the user to work with low dead volume, low fluorescent background, and the device is easily connectable to macro components such as pumps. Cellix’s Mirus Evo Nanopump has also facilitated the execution of the experiment by providing an accurate tool for controlled dispensing or aspiration of liquids at a very stable flow rate within the flow cell, an essential factor for repeatability and consistency of the outcomes.

Figure 8- a) Photograph of Hysens Biochip designed by Mildendo and Cellix; b) Cellix Mirus Evo Nanopump.
Cellix has also designed, manufactured and delivered to University of Valencia a second prototype of a flow cell device, which facilitated the optical detection of intelligent assembly 1. The experiments in UVEG showed promising results using ion-selective organic electrochemical transistors (IS-OECT).

Figure 9- Flow cell and LEC sensor assembly (left). Flow cell and sensor integrated within the chip carrier (right).
A setup capable of the detection of Hg2+ and F- ions using intelligent assembly type 2 has been also demonstrated in microfluidic flow cell by Tyndall and Cellix. The substrates with silver nanoparticles and a gold layer were functionalized with C5 or C8 and then inserted in the HYSENS biochip for the detection which showed promising results (Task 5.4).
During the last three months of the Hysens project, Cellix has closely cooperated with Scriba and UNIBO for the preparation and testing of quantum dots C1 functionalized optical tags sensitive to the presence of sodium ions dissolved in a solution. These optical tags, manufactured by Scriba on a PET / aluminum foil, were integrated into the HYSENS biochips and inserted into a customized microscope frame by Cellix for the optical readout (Task 5.3). The optical readout was recorded using a spectrometer, while the Mirus Evo Nanopump was dispensing solutions having different concentration of ions. The experiments had positive outcomes showing consistency and sensitivity towards detection of sodium chloride in water and in serum (See Figure 10 and Figure 11).

Figure 10 - Image of the optical tag QD-C1 after perfusion with control solution (a), after perfusion with NaCl solution 400ng/L (b), after perfusion with NaCl solution 200ug/L (c)


Figure 11 - Spectrograph showing the peak intensities at 597 nm of the three sensors exposed to the three different saline solutions: control (red), NaCl 400 ng/L (blue), NaCl 200ug/L (purple)
In collaboration with Cellix and Mildendo, UNIBAS-Phys has checked different possibilities to further develop the microfluidic system used for measuring hybrid arrays 4. A new liquid cell design has been created by Cellix and several approaches considered. However, the high level of miniaturization required for the current layout of hybrid arrays 4 and several compatibility issues has led to the conclusion that a PDMS flow cell is the ideal candidate for the targeted application. Moreover, the various discussions led to an improved PDMS flow cell based on 3D microfluidics. (Task 5.5)
The assessment of the reliability and robustness of the HYSENS prototypes has provided exceptional results given the predetermined duration of the project. Also a particular attention has been given to finding more economical alternatives for components and fabrication techniques, thus increasing the cost-effectiveness of the system. The costs achieved for the microfluidic fabrication are relatively inexpensive (now around 10€ per unit) with the possibility of further cost reductions as a scalable technology has been employed.

1.3.6 WP6: Industrial Validation. Leader: Mildendo
The overall goal or milestone of WP6 is to define an exploitation strategy for the outcomes of the HYSENS project. In other words, the consortium needs to define what is the best possible route to exploit or commercialize the resulting prototype product and/or intellectual property and/or know-how from this project.
To do this, there were four main tasks outlined with 5 resulting deliverables:

Deliverables outstanding: none
WP6 conclusions: Creation of Hysens website, Hysens brochures and Hysens animations. Compilation of the mini-business plan at M3, IP strategy plan (D6.2) and an updated business plan at M36, upon end of the project (D6.3). Dissemination of results has taken place during the duration of the project through publications, conference talks, posters, workshops and exhibition at trade shows. As mentioned, final dissemination will take place after the completion of the project in form of a flyer and a press release.
WP6 summary of achievements
Task 6.1. Project website, including "Consortium-only" area.
A website was designed to publicize the HYSENS project, it’s overall technological aims and to disseminate progress of the project to the wider scientific community: http://www.hysens.eu/. This is now one of the key tools used to disseminate information regarding the HYSENS consortium and technology. The website includes details of publications, presentations and tools for exploitation including: Hysens brochures, Hysens animations and flyers. The website was compiled by Cellix and Tyndall-UCC and maintained by Cellix.
Task 6.2. IP strategy plan, M21
The IP strategy plan, an internal report, was written by Cellix and contained information on background IP which may be relevant to the consortium. Some of the industrial inventors listed on background patents include Isis Innovation, Boeing Co., Koninkl Philips Electronics NV, Olympus Optical Co., Samsung Electronics Co. Ltd., Molecular Probes, Xerox Corp. and Motorola Inc. A number of these companies were approached in order to promote HYSENS and investigate possible commercialisation options.
In addition, three patents were filed during the HYSENS project by University of Bologna, Tyndall and University of Valencia. A summary of each patent is listed on the HYSENS website. Technology transfer offices for each university will continue to promote these technologies through their own channels.
Task 6.3. Mini-Business plan for exploitation of project results at M36
The self-explanatory title explains the overall goal of this task which has been submitted .
Recommended strategy to achieve commercialisation of HYSENS technology:
In summary, potential industry partners recommended that the strategy for the HYSENS consortium in demonstration of the biosensors should focus on increasing affordability, portability and accessibility and broadening the spectrum of applications. This could be achieved by:
• Completion of integration and miniaturization of the biosensors.
• Focus on reduced complexity to enhance its accessibility especially in remote places or resource-limited locations.
• Focus on validating chosen applications. Ultimately data needs to be generated to showcase the technology capabilities to potential industry partners; all companies interviewed requested more data on sensors integrated into the microfluidic biochip format.
Commercialization outputs of HYSENS
One of the commercial outputs of the HYSENS project was the fabrication of the HYSENS biochip which can be used as a generic chip in which to embed a variety of different sensors in a microfluidic format for test purposes. This provides a low cost, low risk and effective way of testing a sensor prior to engaging in expensive trials or prototyping of biochips in order to adapt a sensor to a microfluidic format.
Task 6.4. Mini-Business plan for exploitation of project results at M1
Details of the business plan prepared for Deliverable 6.4 can be found in M12, page 27. This plan was further developed as the project progressed and the Deliverable 6.2 (IP port folio and strategy) feed into this.
Task 6.5. Project dissemination.
Tyndall-UCC is in charge of collecting final results of the project which has been organised in a publishable summary folder.
Executive summaries containing details of major achievements including IP and publications from each partner have already been collected by Tyndall-UCC and submitted as deliverable 6.5.
A series of outreach activities have been carried out by Tyndall-UCC and other partners throughout the length of the project.Tyndall was in charge of design of flyers summarizing main results obtained in the project. Two flyers were designed, one for general use and one for specialised use. Tyndall also wrote press release at the beginning of the project to promote the launch of Hysens activities.
Project Results:
Potential impact
The aim of the European Unions’ Framework Programme call for which HYSENS is associated is focused on the progression of technologies in the fields of nanosciences, nanotechnologies, materials and new production technologies, ideally enabling industrial partners to develop new technology products and markets. In order to achieve this aim the HYSENS project has developed a prototype generic biochip to embed a number of different biosensors which were developed during the project.
In order to investigate a potential commercialisation strategy for the HYSENS technology, a number of tasks were executed. These included a mini-business plan with a study of the biosensors market and the blood and gas electrolyte market (applications for which the HYSENS sensors were targeting). This market study was consolidated with feedback from interviews that Cellix conducted with industry stakeholders. Results of the HYSENS project and the maturity of the technology in general was assessed and compared against technologies currently available on the market. As a result of this strategic analysis, a number of recommendations for future exploitation of the HYSENS technology were made, including focusing on increasing affordability, portability and accessibility and broadening the spectrum of applications. This could be achieved by:
• Completion of integration and miniaturization of the biosensors.
• Focus on reduced complexity to enhance its accessibility especially in remote places or resource-limited locations.
• Focus on validating chosen applications. Ultimately data needs to be generated to showcase the technology capabilities to potential industry partners; all companies requested more data on sensors integrated into the microfluidic biochip format.
A positive benefit of integrating HYSENS sensor in the microfluidic biochip format, was the skill and expertise which Cellix developed in this area. As a result, there is a very realistic exploitation path for the SMEs in the HYSENS consortium to offer the “HYSENS biochip” as a generic test chip for diagnostic companies seeking to transition their current tests on bulky clinical analyzers to microfluidic formats which could enable them to target point-of-care markets. The SMEs may offer this in the context of an OEM partnership.
Socio-economic impact
Socio-economic impacts are directly related to the future commercial exploitation and increase of employment in countries where the SMEs are placed. Societal implications are also expected improving the enforcement of EU legislations and having an impact in the health field.
Diagnostic assays are becoming increasingly important in a wide variety of fields, from testing water quality to diagnosis of a disease. The days of the block-buster, “one-size-fits-all” drugs are waning as there is an ever-increasing focus on personalized medicine and the delivery of therapies tailored to a specific individuals’ condition. It therefore follows that we must deliver technology that is capable of executing these diagnostic assays. The HYSENS consortium has focused on delivering sensors which are inexpensive, more sensitive and selective, and embedded in microfluidic biochips, meaning they may be adapted easily to point-of-care devices.
While the HYSENS sensors may not be ready for market at this point, the SMEs within the HYSENS consortium have demonstrated a strong competence, and acquired further experience in the design and manufacture of microfluidic devices with respect to embedding sensors on-chip and this expertise is one which may be offered as service, in an OEM capacity, to diagnostic companies.
One of the commercial outputs of the HYSENS project was the fabrication of the HYSENS biochip which can be used as a generic chip in which to embed a variety of different sensors in a microfluidic format for test purposes. This provides a low cost, low risk and effective way of testing a sensor prior to engaging in expensive trials or prototyping of biochips in order to adapt a sensor to a microfluidic format.
Cellix exhibited details of the HYSENS project at the Lab-on-a-chip congresses in San Diego (September 2013) and Berlin (March 2014). As a result, Cellix has received interest from diagnostic companies who wish to embed their sensors on-chip with a view to targeting point-of-care markets. At present, Cellix has engaged with one diagnostic company (whose revenues exceed €100M) and have used the HYSENS biochip as a test-chip in which to embed their sensors. Should this prove successful, Cellix will co-develop a new biochip with this company where expected units for year one (2015) are 1,000-10,000 biochips.
In order to further capitalize on this, Cellix has highlighted on the HYSENS website how companies can engage with Cellix to test their sensors on-chip. We will continue to promote this at future conferences and trade shows including the “Siemens – Future of in vitro Diagnostics” conference in Palo Alto, April 2014 where Cellix has been invited to showcase their technology and will include the HYSENS biochip.
This has a knock-on effect for other SMEs within the HYSENS consortium. Mildendo fabricated the microfluidic biochip which Cellix is using to engage various diagnostics companies. Cellix’s success in this area will also have a positive effect of increased sales for this biochip for Mildendo.
Overall, the benefit of SMEs such as Cellix and Mildendo, being involved in the HYSENS project is an increased skill-set and further understanding of the diagnostics market which will ultimately result in them addressing wider markets and gaining higher revenues. Successful exploitation of this innovative and competitive positioning within this market sector will generate employment throughout the supply chain: chip design, prototype fabrication and testing, manufacturing, quality control and testing.
Societal implications
The development of more sensitive and selective sensors which are available at a lower price will bring considerable benefits, not only to the wider research community but also in applications for point-of-care use.
Main dissemination activities
Furthermore, project logo, diagrams or photographs illustrating and promoting the work of the project (including videos, etc…), as well as the list of all beneficiaries with the corresponding contact names can be submitted without any restriction.

Potential Impact:
www.hysens.eu
The HYSENS website was one of the key tools used to disseminate details of the project, it’s overall technological aims and to disseminate progress of the project to the wider scientific community. The website includes details of publications, presentations (oral and poster), exhibitions.

List of Websites:
1.1 Address of public website
www.hysens.eu
The HYSENS website was one of the key tools used to disseminate details of the project, it’s overall technological aims and to disseminate progress of the project to the wider scientific community. The website includes details of publications, presentations (oral and poster), exhibitions.
Some snapshots of the website are highlighted below:






The website also includes tools for exploitation including:
o HYSENS Brochures: Three brochures were designed during the project. The first brochure was designed as a short overview of the HYSENS project.




Brochure 1: HYSENS Overview

Two further brochures were developed towards the end of the project; one for dissemination to the scientific community publicising results of the HYSENS project and a second one for the general public giving an overview of the importance of the results achieved to the wider community.

Brochure 2: HYSENS Scientific Results


Brochure 3: HYSENS General Flyer for wider public dissemination

All brochures are downloadable as pdfs from the website and can be used by partners to explain the project to potential interested partners (industry, academia, press etc.).
o HYSENS animation: This is short animation which briefly explains how an assembly, embedded on-chip, is used as a sensor for the detection of analytes. This is hosted on Cellix’s YouTube and linked directly to the HYSENS website homepage. A second animation was produced from July – September 2013 to explain the operation of the nanowire embedded sensor from University of Basel. As with the first animation, this is also hosted on Cellix’s YouTube channel and linked directly to the HYSENS website homepage. Both animations have been shown by Cellix at two exhibitions; Lab-on-a-chip World Congress, San Diego, September 2013 and Lab-on-a-chip European Congress, Berlin, March 2014.

Animation explaining HYSENS sensor embedded in microfluidic biochip


Close-up of video animation explaining the embedded sensor on-chip.