Leader: Paolo Matteini (CNR-IFAC); Other collaborator(s): Aloisi, Capone, Forleo (CNR-IMM); Baldini, Berneschi, D'Andrea, Matteini (CNR-IFAC)
Specific studies aimed at advancing established and emerging analytical techniques toward the identification of characteristic biomarker patterns in diverse human matrices will be pursued. A first activity concerns the setup of platforms based on Raman and on fluorescence techniques for capturing the optical fingerprint and for multianalyte detection in real samples. Another activity focuses on volatilomics through the development of an electronic nose trained by GC/MS volatilome data and the optimization of a Solid Phase Microextraction-GC/MS method for analyzing volatile organic compounds profiles in liquid biosamples. As complementary activity a sweat collection tool based on the integration of microchannels, reservoirs, and nano-porous textile sponges will be developed.
Brief description of the activities and of the intermediate results
The activity involves the development of a surface-enhanced Raman scattering (SERS) sensing platform for optical fingerprinting of biological fluids, including serum. Nanostructured silver spotted, flexible and disposable substrates are under development to generate an optical enhancement in the Raman signal and facilitating the use of small biofluid aliquots according to a time-efficient procedure. Another activity centres on an optical platform utilizing long period gratings (LPGs) for biomarker sensing. The platform feature multianalyte detection using different LPGs on a single fiber, each with distinct resonance wavelengths and containing a unique biological recognition element selective to a specific biomarker. About the volatilomics activity, the SPYROX electronic nose device development is ongoing, utilizing chemoresistive gas sensors trained by volatilome data obtained through GC/MS. SPYROX serves as a digital fingerprinting tool for the volatilome of selected biofluids. As complementary acivity, the exploration of cutting-edge technologies and materials for skin-interfaced sweat collection systems. This involves the integration of microchannels, reservoirs, and a combination of nano-porous textile sponges to facilitate sweat collection during on-body wear. These activities can support intervention study of WP1 (In-Tempo) on a subpopulation and that of WP3 (I COUNT) on the total population for secondary explorative objectives.
Main policy, industrial and scientific implications
Promotion of interdisciplinary research: The proposed activity may call for funding policies that encourage collaboration across diverse fields (biochemistry, engineering, nanotechnology), fostering the development of emerging technologies like SERS devices and wearable sweat collection systems;
Growth in diagnostic device markets: The introduction of advanced platforms like that based on SERS and SPYROX could drive innovation in wearable and portable diagnostic tools, opening new market opportunities in healthcare technology.
Enhanced biomarker detection
The use of multi-analyte platforms (SERS, LPG) enables more precise and simultaneous detection of biomarkers, advancing research in early disease diagnosis and personalized medicine
The proposed platforms are at an advanced stage of development, undergoing validation tests or early-stage applications in clinical research settings.
SERS platform: The development of nanostructured silver substrates has been finalized, optimizing their performance for enhanced optical fingerprinting. The SERS platform has been tested on a wider range of biomolecules (HSA, BSA, Lysozime, Tranferrrin, Amyloid b42. etc.) to validate its effective and sensitive detection capabilities.
LPG sensor: The multianalyte detection capabilities of LPG sensors are in progress by developing the algorithm to extract the information derived by the signal modulation caused by the interaction of the detected molecules with the different sensing biolayers deposited on the LPGs. In parallel the microfluidic circuit associated with the flow cell containing the fibre with the inscribed LPGs on it has been further developed using suitable syringe pumps and 3-way valves. The developed platform will be tested on some of the biomolecules already tested on the SERS platform.
SPYROX electronic nose and GC/MS analysis: The development of an electronic nose devoted to human volatilome fingerprinting was completed. The device, code-named SPYROX, adopts an array of 8 metal-oxide (MOX) gas sensors and it can analyze response signals from different matrices (multi-matrix samples), dealing with exhaled breath and headspace analysis of human biological samples. The functionality of the device was proved by a classification test of chemical standards and VOC mixtures as well. The device is currently being used in the analysis of blood and urine jointly with volatilome analysis by SPME-GC/MS on a sample population within the framework of scientific collaborations.
Sweat collection tool: a patch-based multilayer system has been designed and medical-grade polymeric films with hydrophilic/hydrophobic properties are now under consideration to increase the volume of collected sweat in a leakage-free manner.
The proposed platforms underwent further optimization improving their use in real settings.
SERS platform: The SERS platform has been further optimized in the form of a wearable SERS-active chip by the use of biocompatible and transparent adhesive tapes adhered on each side of the substrate and proper modification with a small conduct allowing the direct flow of the biofluid (such as sweat) to the sensor.
LPG sensor: The activity dedicated to determining the algorithm to extract data from two LPGs written along the same fibre in order to allow the simultaneous measurement of two analytes is still ongoing; the flow-cell is ready to be used for the determination, at the moment, of a single analyte.
SPYROX electronic nose and GC/MS analysis: The electronic nose has been further configured for fingerprinting the human volatilome based on the headspace analysis of human biological samples (as urine, blood and exhaled breath collected in Tedlar gas sampling bag). The activity of using the device in the analysis of human biological samples, jointly with the analysis of volatiles by SPME-GC/MS, is still ongoing; urine samples have been made available within scientific collaborations external to Age.it, awaiting the biological samples that will be collected in the In-Tempo study protocol in WP1 and in the ICount study protocol in WP3 for secondary exploratory objectives.
Sweat collection tool: natural polysaccharide blends (including alginate and chitosan) have been tested in the patch integrated microfluidic channel and the characterization is still ongoing.
The proposed platforms underwent further optimization improving their use in real settings.
SERS platform: The SERS platform has been further tested against molecular standards as urea and lactic acid.
LPG sensor: The activity dedicated to determining the algorithm to extract data from two LPGs written along the same fibre in order to allow the simultaneous measurement of two analytes is still ongoing; the flow-cell has been tested for the detection, at the moment, of a single analyte (FKBP12 protein involved in the variety of cancer pathologies).
SPYROX electronic nose and GC/MS analysis: The experimental activity with the electronic nose based on arrays of gas sensors for the fingerprint of the human volatilome in biological samples such as urine, blood and exhaled breath has continued. In conjunction with the volatilome characterization with the device, the analysis of human biological samples by SPME-GC/MS is carried out. The biological samples (urine and whole blood) were collected on a sample subpopulation within the In-Tempo study protocol in WP1. The development of the experimental protocols led to a publication with the other partners of WP1. In the meantime, urine samples have been made available by scientific collaborations external to Age.it, and in this current reference period the analysis campaign on this set of biosamples with the related data analysis is underway.
Sweat collection tool: FIB technology has been exploited to fabricate reproducible micro- and nanoscale cuts on PET medical grade adhesive membrane.
The proposed platforms have undergone further optimization, enhancing their applicability in real-world scenarios.
SERS platform: The SERS platform was succesfully tested against urea and lactic acid, as biomarkers of different diseases (including aging-related) found in sweat (as well as in blood). Firstly it was calibrated using a simulated sweat solution, providing a linear response in the 4–250 mM and 1.67–250 mM ranges for urea and lactate, respectively. As a proof of validation of the platform, we also performed experiments using real sweat samples produced by a volunteer during physical exercise. Analysing the sweat from different rounds of physical activity, we detected lactate and urea concentrations ranging from 19 to 60 mM and from 50 to 80 mM, respectively.
LPG sensor: The algorithm to extract data from two LPGs written along the same fibre in order to allow the simultaneous measurement of two analytes was determined. It involves the use of two LPGs coupled in the same cladding mode (Mach-Zehnder configuration). In this configuration, the light launched in the fiber's core mode is coupled into the cladding mode via the first LPG and then subsequently recoupled into the core mode via the second LPG. Just as with the classic free-space Mach-Zehnder interferometer, the difference in optical pathlengths between the core and cladding paths produces interference, and the fiber's output spectrum is composed of interference fringes rather than isolated peaks.
SPYROX electronic nose and GC/MS analysis: Experimental work continued using the SPYROX electronic nose, which employs arrays of gas sensors to capture the unique fingerprint of the human volatilome in biological samples such as urine, blood, and exhaled breath. Alongside this, human biological samples are also being analyzed using SPME-GC/MS techniques for comprehensive volatilome characterization. Urine and whole blood samples were collected from a subset of participants under the In-Tempo study protocol in WP1. These samples have been stored frozen and will be shipped in batches once the collection phase is complete. The development of experimental protocols has resulted in a joint publication with other WP1 partners. Unfortunately, the ICount study protocol in WP3, which was expected to provide additional biosamples, faced implementation challenges. In the meantime, urine samples have been obtained through external scientific collaborations outside of Age.it. During the current reporting period, the analysis campaign on this set of biological samples has been concluded with the related data analysis.
Scientific publications:
Conferences: