Fluorescence-based sensing
This activity is focused on the design, development and characterization of fluorescence based sensors and on novel fluorescence-based optical platforms and devices and of optical chips for the simultaneous detection of more than one analyte and the continuous monitoring of chemicals and biochemicals.
The main applications of the realized devices have been in the biomedical field and measurements were performed on drawn serum/plasma samples or on dialysate by connecting the device to the human body by means of microdialysis.
In particular, fluorescence-based devices for point of care testing (POCT) have been designed and implemented. In the recent years, a demanding request has been raised by physicians for the development of devices to be located close to the patient’s bed and able to provide in a short time the measurement of bioanalytes of interest, the so called POCT device. This request derives from the necessity to perform shortly the measurement of bioanalytes, the knowledge of which can help the physicians to formulate the right diagnosis or to decide the correct therapy, avoiding to deliver the samples to the central laboratories and to wait, for hours, for the results of the analysis. In general, in POCT application, multianalyte detection of a few biomarkers is required, differently from genomics or proteomics. The last years have seen an incredible effort led by research centers and industries in the design and development of POCT devices, with fertility tests in urine, glucose in adipose tissue and measurement of blood gases and electrolytes being the first area of applications. If blood gas and electrolytes are excluded, most of the POCT devices on the market are able to measure one analyte per time, and - in the case of multiple detections - they generally make use of separate cartridges. The combination of optical biochip and microfluidics can offer the possibility of parallelization with the simultaneous measurement of different analytes at the same time on the same chip.
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Activity is focused on the design, development and characterisation of novel optical platforms and devices and of optical chips for the simultaneous detection of more than one analytes and the continuous monitoring of chemicals and biochemicals. Measurements are performed on drawn serum/plasma samples or connecting the device to the human body by means of microdialysis.
As for the analytes, the activity is related on the determination of:
- BIOMARKERS FOR SEPSIS
(CRP, procalcitonin, interleukine-6,neopterin, suPAR);
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- IMMUNOSUPPRESSANTS IN TRANSPLANTED PATIENTS
(cyclosporin A, mycophenolic acid, tacrolimus)
- TRANSCRIPTION FACTOR NF-kB
A collaboration with the Institute of Photonics of the Duke University (Vo-Dinh) led to the development of optical sensors for the detection of the transcription factor NF-kB and to the development of optical fibre tip-based sensors using molecular beacons for the detection of mRNA encoding survivin.
Projects
NANODEM (“NANOphotonic DEvice for Multiple therapeutic drug monitoring”)
CAREMAN (“HealthCARE by Biosensor Measurements And Networking”)
ARTEMIDE (“Autonomous Real Time Embedded Multi-analyte Integrated Detection Environment”)
Main Publications on the subject
C. Berrettoni, S. Berneschi, R. Bernini, A. Giannetti, I.A. Grimaldi, G. Persichetti, G. Testa, S. Tombelli, C. Trono, F. Baldini: Optical monitoring of therapeutic drugs with a novel fluorescence-based POCT device, Procedia Engineering, 87, 392-395, 2014.
Abstract :A novel optical biochip for immunosuppressants detection in transplanted patients is described. The optical working of the chip, based on total internal reflection fluorescence and consisting of two bonded polymeric parts, a Zeonex 330 slide and a Topas foil was demonstrated. In parallel, a heterogeneous competitive immunoassay for tacrolimus was implemented using a thirteen microchannel optical chip interrogated by an in-house optical platform based on fluorescence anisotropy and the bioassay conditions were optimized: a limit of detection of 0.11 ng/mL, a limit of quantification of 0.57 ng/mL and a coefficient of variation of 25% were achieved.
F. Baldini: Microdialysis-based sensing in clinical applications, Anal. Bioanal. Chem., 397, pp. 909–916, 2010.
Abstract:The need for fast and continuous measurements in the biomedical field is driving scientists to look for an alternative to blood sampling. This implies the adoption of invasive approaches, which, in some cases, may lead to reduced safety for the patient; consequently this strategy is pursued only if it is unavoidable. Microdialysis-based sensing provides a minimally invasive solution, with biological samples drawn by means of a microdialysis catheter and examined outside the human body. Therefore, it has become a promising approach to investigate the interstitial fluid in human brain and subcutaneous adipose tissue, providing important information on the tissue biochemistry and metabolism. Advantages and limitations of microdialysis are considered here and the applications in the clinical field are described, with the provision of some examples and with a view to the new perspectives in the field.
F. Baldini, A. Carloni, A. Giannetti, G. Porro, C. Trono: An optical PMMA biochip based on fluorescence anisotropy: application to C–reactive protein assay, Sensors and Actuators B, 139, pp. 64–68, 2009
Abstract:An optical platform for point of care testing (POCT) application is developed. The core of the platform is a miniaturised polymethylmetacrylate (PMMA) chip, that consists of two pieces of PMMA that have been custom-machined to produce flow channels, 500m in width and 400m in height, through which the analysed sample flows. Thanks to the fluorescence anisotropy, a large fraction of the fluorescence emitted by the sensing layer immobilized on the bottom of the PMMA cover travels along the thickness of the PMMA cover itself up to its end-face and it is collected by a plastic optical fibre connected to a photodetector. The potential of the optical chip is investigated with a sandwich assay for the C-reactive protein, one of the markers for inflammatory diseases. The assay covers the working range from 0.1 to 50mgL−1 with a limit of detection of 0.004mgL−1. Different combinations of capture and target antibodies were carefully investigated in order to achieve the best performances in terms of sensitivity and unspecific adsorption.
F. Baldini, L. Bolzoni, A. Giannetti, M. Kess, P. M. Krämer, E. Kremmer, G. Porro, F. Senesi, C. Trono: A new procalcitonin optical immunosensor for POCT applications, Anal. Bioanal. Chem., 393, pp. 1183–1190, 2009
Abstract:A new immunosensor for the determination of procalcitonin was developed. A sandwich assay format was implemented on a polymethylmetacrylate optical biochip, opportunely shaped in order to obtain several flow channels and potentially suitable for point of care testing applications. The sandwich format makes use of two new rat monoclonal antibodies. The capture antibody was covalently immobilised on the surface of the plastic chip, and the detection antibody was labelled with DY647 dye. Different combinations of capture and detection antibodies were investigated, and particular attention was devoted in order to avoid the nonspecific adsorption. A limit of detection of 0.088 mg L−1 was achieved within the working range of 0.28–50 mg L−1 in buffer samples. The assay was also implemented in human serum, and 0.2 and 0.7–25 mg L−1 were the attained limit of detection and working range, respectively.
F. Baldini, A. Carloni, A. Giannetti, A. Mencaglia, G. Porro, L.Tedeschi, C. Trono: Optical PMMA chip suitable for multianalyte detection, IEEE Sensors Journal, 8 (4), pp.1305-1309, 2008
Abstract:A new optical platform for the interrogation of a polymethylmethacrylate (PMMA) multichannel array for chemical and biochemical parameters is described. It consists of a plastic chip formed by two pieces of PMMA properly shaped in order to obtain different microchannels, 500 m in width and 400 min height. A fluorescent sensing layer is immobilized on the internal wall of the microchannel. The emitted light travels along the thickness of the upper PMMA piece and it is detected with an optical fiber connected with a spectrum analyzer. The anisotropy of the fluorescence emitted by the fluorophore immobilized at the liquid/plastic interface gives rise to preferential directions of the emitted fluorescence. The collection of the fluorescence at an angle of 50 implies an amplification of roughly one order of magnitude in the ratio between the fluorescence signal and the scattered light. The optical platform was further characterized as a pH sensor and as potential chip for immunoassay. In the first case, the fluorescein dye is directly immobilized onto the internal wall of the channel and the fluorescence changes are measured as a function of the pH of the flowing sample. In the second case, a direct antigen–antibody interaction is carried out. The mouse-IgG is covalently immobilized onto the internal wall of the channel and the Cy5-labeled anti-mouse IgG is used for the specific interaction.
F. Baldini, A. Carloni, A. Giannetti, G. Porro, C. Trono: A new optical platform for biosensing based on fluorescence anisotropy, Anal. Bioanal. Chem., 391, pp. 1837–1844, 2008
Abstract:A novel fluorescence-based optical platform for the interrogation of an optical biochip was designed and developed. The optical biochip was made of poly(methyl methacrylate) (PMMA) formed by two pieces of PMMA appropriately shaped in order to obtain four microchannels that are 500-μm wide and 400-μm high. The lower part includes the microchannels and the inlet and outlet for the fluidics, while the sensing biolayer was immobilized on the upper part. The optical signal comprised the fluorescence emitted by the biolayer, which was anisotropically coupled to the PMMA cover and suitably guided by the PMMA chip. The potentiality of the optical chip as a biosensor was investigated by means of a direct IgG/anti-IgG interaction carried out inside the flow channels. The mouse-IgG was covalently immobilized on the internal wall of the PMMA cover, and the Cy5-labelled anti-mouse IgG was used for the specific interaction. Several chemical treatments of the PMMA surface were investigated, poly(L-lactic acid), Eudragit L100 and NaOH, in order to obtain the most effective distribution of carboxylic groups useful for the covalent immobilisation of the mouse-IgG. The treatment with Eudragit L100 was found to be the most successful. Limits of detection and quantification of 0.05 μg mL−1 and 0.2 μg mL−1, respectively, were obtained with the configuration described.
