Heterogeneous Integration of Acoustic Microextraction with an Optoelectronic Sensor on Glass for Nucleic Acid Testing.

Lab-on-a-chip systems (LOCs), characterized by their high sensitivity, low sample consumption, and portability, have significantly advanced the field of on-site testing. Despite the evolution of integrated LOCs from qualitative to quantitative analyses, on-chip full integration of sample preparation, purification, and multiplexed detection remains a challenge. Here, we propose a strategy for the heterogeneous integration of a set of complementary metal oxide semiconductor-compatible devices including acoustic resonator, thin-film resistors, and temperature/photosensors as a new type of LOC for nucleic acid testing (NAT). Programmed acoustic streaming-based particles and fluid manipulations largely simplify the nucleic acid extraction process including cell lysis, nucleic acid capture, and elution. The design of the acoustic microextraction module and extraction process was thoroughly studied. Benefitted by the microelectromechanical system approach, the conventional mechanical actions and complex flow control are avoided, which enables a compact hand-held NAT instrument without complicated peripherals. Validation experiments conducted on plasma-harboring mutations in the epidermal growth factor receptor (EGFR) gene confirmed the robustness of the system, achieving an impressive nucleic acid (NA) extraction efficiency of approximately 90% within 5 min and a limit of detection of the target NA in the plasma of 1 copy/µL.

Chitosan-Coated Fosetyl-Al Nanocrystals' Efficacy on Nicotiana tabacum Colonized by Xylella fastidiosa.

Xylella fastidiosa (Xf) is a quarantine plant pathogen capable of colonizing the xylem of a wide range of hosts. Currently, there is no cure able to eliminate the pathogen from a diseased plant, but several integrated strategies have been implemented for containing the spread of Xf. Nanotechnology represents an innovative strategy based on the possibility of maximizing the potential antibacterial activity by increasing the surface-to-volume ratio of nanoscale formulations. Nanoparticles based on chitosan and/or fosetyl-Al have shown different in vitro antibacterial efficacy against Xf subsp. fastidiosa (Xff) and pauca (Xfp). This work demonstrated the uptake of chitosan-coated fosetyl-Al nanocrystals (CH-nanoFos) by roots and their localization in the stems and leaves of Olea europaea plants. Additionally, the antibacterial activity of fosetyl-Al, nano-fosetyl, nano-chitosan, and CH-nanoFos was tested on Nicotiana tabacum cultivar SR1 (Petite Havana) inoculated with Xff, Xfp, or Xf subsp. multiplex (Xfm). The bacterial load was evaluated with qPCR, and the results showed that CH-nanoFos was the only treatment able to reduce the colonization of Xff, Xfm, and Xfp in tobacco plants. Additionally, the area under the disease progress curve, used to assess symptom development in tobacco plants inoculated with Xff, Xfm, and Xfp and treated with CH-nanoFos, showed a reduction in symptom development. Furthermore, the twitching assay and bacterial growth under microfluidic conditions confirmed the antibacterial activity of CH-nanoFos.

Biomolecular Monitoring Tool Based on Lab-on-Chip for Virus Detection.

Lab-on-Chip (LoC) devices for performing real-time PCR are advantageous compared to standard equipment since these systems allow to conduct in-field quick analysis. The development of LoCs, where the components for performing the nucleic acid amplification are all integrated, can be an issue. In this work, we present a LoC-PCR device where thermalization, temperature control and detection elements are all integrated on a single glass substrate named System-on-Glass (SoG) obtained using metal thin-film deposition. By using a microwell plate optically coupled with the SoG, real-time reverse transcriptase PCR of RNA extracted from both a plant and human virus has been carried out in the developed LoC-PCR device. The limit of detection and time of analysis for the detection of the two viruses by using the LoC-PCR were compared with those achieved by standard equipment. The results showed that the two systems can detect the same concentration of RNA; however, the LoC-PCR performs the analysis in half of the time compared to the standard thermocycler, with the advantage of the portability, leading to a point-of-care device for several diagnostic applications.

Analysis of Italian isolates of Pantoea stewartii subsp. stewartii and development of a real-time PCR-based diagnostic method.

Pantoea stewartii subsp. stewartii (Pss) causes Stewart's vascular wilt of maize, and it is responsible for serious crop losses. Pss is indigenous to North America and spreads with maize seeds. The presence of Pss has been notified in Italy since 2015. The risk assessment of the entry of Pss in the EU from the United States through seed trade is in the order of magnitude of hundred introductions per year. Several molecular or serological tests were developed for the detection of Pss and used as official analysis for the certification of commercial seeds. However, some of these tests lack adequate specificity, not allowing to correctly discriminate Pss from P. stewartii subsp. indologenes (Psi). Psi is occasionally present in maize seeds and is avirulent for maize. In this study, several Italian isolates of Pss recovered in 2015 and 2018 have been characterized by molecular, biochemical, and pathogenicity tests; moreover, their genomes have been assembled through MinION and Illumina-sequencing procedures. Genomic analysis reveals multiple introgression events. Exploiting these results, a new primer combination has been defined and verified by real-time PCR, allowing the development of a specific molecular test able to detect the presence of Pss down to the concentration of 103 CFU/ml in spiked samples of maize seed extracts. Due to the high analytical sensitivity and specificity achieved with this test, the detection of Pss has been improved disentangling the inconclusive results in Pss maize seed diagnosis, overcoming its misidentification in place of Psi. Altogether, this test addresses the critical issue associated with maize seeds imported from regions where Stewart's disease is endemic.

Thin-Film-Based Multifunctional System for Optical Detection and Thermal Treatment of Biological Samples.

In this work, we present a multifunctional Lab-on-Chip (LoC) platform based on hydrogenated amorphous silicon sensors suitable for a wide range of application in the fields of biochemical and food quality control analysis. The proposed system includes a LoC fabricated on a 5 cm × 5 cm glass substrate and a set of electronic boards for controlling the LoC functionalities. The presented Lab-on-Chip comprises light and temperature sensors, a thin film resistor acting as a heating source, and an optional thin film interferential filter suitable for fluorescence analysis. The developed electronics allows to control the thin film heater, a light source for fluorescence and absorption measurements, and the photosensors to acquire luminescent signals. All these modules are enclosed in a black metal box ensuring the portability of the whole platform. System performances have been evaluated in terms of sensor optical performances and thermal control achievements. For optical sensors, we have found a minimum number of detectable photons of 8 × 104 s-1·cm-2 at room temperature, 1.6 × 106 s-1·cm-2 in presence of fluorescence excitation source, and 2.4 × 106 s-1·cm-2 at 90 °C. From a thermal management point of view, we have obtained heating and cooling rates both equal to 2.2 °C/s, and a temperature sensor sensitivity of about 3 mV/°C even in presence of light. The achieved performances demonstrate the possibility to simultaneously use all integrated sensors and actuators, making promising the presented platform for a wide range of application fields.

Split Aptamers Immobilized on Polymer Brushes Integrated in a Lab-on-Chip System Based on an Array of Amorphous Silicon Photosensors: A Novel Sensor Assay.

Innovative materials for the integration of aptamers in Lab-on-Chip systems are important for the development of miniaturized portable devices in the field of health-care and diagnostics. Herein we highlight a general method to tailor an aptamer sequence in two subunits that are randomly immobilized into a layer of polymer brushes grown on the internal surface of microfluidic channels, optically aligned with an array of amorphous silicon photosensors for the detection of fluorescence. Our approach relies on the use of split aptamer sequences maintaining their binding affinity to the target molecule. After binding the target molecule, the fragments, separately immobilized to the brush layer, form an assembled structure that in presence of a "light switching" complex [Ru(phen)2(dppz)]2+, emit a fluorescent signal detected by the photosensors positioned underneath. The fluorescent intensity is proportional to the concentration of the target molecule. As proof of principle, we selected fragments derived from an aptamer sequence with binding affinity towards ATP. Using this assay, a limit of detection down to 0.9 µM ATP has been achieved. The sensitivity is compared with an assay where the original aptamer sequence is used. The possibility to re-use both the aptamer assays for several times is demonstrated.

The Hydroxytyrosol Induces the Death for Apoptosis of Human Melanoma Cells.

Melanoma is the most aggressive form of skin cancer and one of the most treatment-refractory malignancies. In metastatic melanoma cell lines, we analysed the anti-proliferative and pro-apoptotic potentials of a phenolic component of olive oil, the hydroxytyrosol. In particular, through MTS assay, DeadEnd™ Colorimetric TUNEL assay, Annexin V binding and PI uptake, western blot experiment, intracellular reactive oxygen species (ROS) analysis, and the cell colony assay, we showed that the hydroxytyrosol treatment remarkably reduces the cell viability inducing the death for apoptosis of melanoma cells. Moreover, we showed that the hydroxytyrosol treatment of melanoma cells leads to a significant increase of p53 and γH2AX expression, a significant decrease of AKT expression and the inhibition of cell colony formation ability. Finally, we propose that the increased amount of intracellular reactive oxygen species (ROS) that may be related to the regulation of the pathways involved in the activation of apoptosis and in the inhibition of melanoma growth could be the strategy used by hydroxytyrosol to exert its functions in melanoma. Therefore, for its role in melanoma growth inhibition, the hydroxytyrosol treatment could deeply interfere with melanoma progression as a promising therapeutic option for the treatment of this highly invasive tumour.

Dibutyltin(IV) and Tributyltin(IV) Derivatives of meso-Tetra(4-sulfonatophenyl)porphine Inhibit the Growth and the Migration of Human Melanoma Cells.

Melanoma is the most aggressive and deadly form of skin cancer, which is largely due to its propensity to metastasize. Therefore, with the aim to inhibit the growth and the metastatic dissemination of melanoma cells and to provide a novel treatment option, we studied the effects of the melanoma treatment with two organotin(IV) complexes of the meso-tetra(4-sulfonato-phenyl)porphine, namely (Bu2Sn)2TPPS and (Bu3Sn)4TPPS. In particular, we showed that nanomolar concentrations of (Bu2Sn)2TPPS and (Bu3Sn)4TPPS are sufficient to inhibit melanoma cell growth, to increase the expression of the full-length poly (ADP-ribose) polymerase (PARP-1), to induce the cell cycle arrest respectively at G2/M and G0/G1 through the inhibition of the Cyclin D1 expression and to inhibit cell colony formation. Nanomolar concentrations of (Bu2Sn)2TPPS and (Bu3Sn)4TPPS are also sufficient to inhibit the melanoma cell migration and the expression of some adhesion receptors. Moreover, we report that (Bu2Sn)2TPPS and (Bu3Sn)4TPPS act downstream of BRAF, mainly bypassing its functions, but targeting the STAT3 signalling protein. Finally, these results suggest that (Bu2Sn)2TPPS and (Bu3Sn)4TPPS may be effective therapeutic strategies for their role in the inhibition of melanoma growth and migration.

Fluorescent Label-Free Aptasensor Integrated in a Lab-on-Chip System for the Detection of Ochratoxin A in Beer and Wheat.

This paper reports on the development of a fluorescent label-free aptamer assay integrated in a lab-on-chip (LoC) system for the detection of Ochratoxin A (OTA). The detection system relies on the integration, on a single glass substrate, of an array of amorphous silicon photosensors and a long pass interferential filter. The aptamer assay, integrated into the microfluidic network, is an aptasensor having affinity versus OTA, selected as a case study. The fluorescent molecule is a "light switch" complex [Ru(phen)2(dppz)]2+. The aptamer is directly anchored into a layer of poly(2-hydroxyethyl methacrylate) polymer brushes grown inside the channels. The fluorophore is intercalated between the base pairs of the aptamer. Upon the interaction of OTA with the aptasensor, a change of the aptamer conformation causes the release of the fluorophore, yielding a decrease of the fluorescent signal detected by the array of the amorphous silicon photosensors positioned underneath the microfluidic network. The developed LoC is a portable system capable of performing the analysis with a small volume of sample (about 10 µL) in a short time (5 min) with a limit of detection for OTA equal to 1.3 ng/mL. The LoC has been applied for the detection of OTA (5-200 ng/mL) in beer and wheat samples.

Integrated chemiluminescence-based lab-on-chip for detection of life markers in extraterrestrial environments.

The detection of life markers is a high priority task in the exploration of the Solar System. Biochips performing in-situ multiplex immunoassays are a very promising approach alternative to gas chromatography coupled with mass spectrometry. As part of the PLEIADES project, we present the development of a chemiluminescence-based, highly integrated analytical platform for the detection of biomarkers outside of the Earth. The PLEIADES device goes beyond the current lab-on-chip approaches that still require bulky external instrumentation for their operation. It exploits an autonomous capillary force-driven microfluidic network, an array of thin-film hydrogenated amorphous silicon photosensors, and chemiluminescence bioassays to provide highly sensitive analyte detection in a very simple and compact configuration. Adenosine triphosphate was selected as the target life marker. Three bioassay formats have been developed, namely (a) a bioluminescence assay exploiting a luciferase mutant with enhanced thermal and pH stability and (b and c) binding assays exploiting antibodies or functional nucleic acids (aptamers) as biospecific recognition elements and peroxidase or DNAzymes as chemiluminescence reporters. Preliminary results, showing limits of detection in the nanomolar range, confirm the validity of the proposed approach.

Integrated Optoelectronic Device for Detection of Fluorescent Molecules.

This paper presents the development of a compact optoelectronic device suitable for on-chip detection of fluorescent molecules. In order to obtain a highly integrated device, a long-pass multi-dielectric filter has been integrated with thin-film amorphous silicon photosensors on a single glass substrate. Filter rejects the excitation light, allowing the reduction of the distance between the source and the fluorescent site and avoiding the use of external optical component. The compatibility of the technological processes determined the materials and the temporal sequence of the device fabrication. The developed device has been designed for the fluorescence detection of ruthenium complex based molecules and tested, as a proof of concept, for the detection of double-stranded DNA down to 0.5 ng. Results demonstrate the correct operation of the integrated system in both rejecting the excitation light and in detecting the fluorescent signal, demonstrating the suitability of this optoelectronic platform in practical biomedical applications.

An All-Glass Microfluidic Network with Integrated Amorphous Silicon Photosensors for on-Chip Monitoring of Enzymatic Biochemical Assay.

A lab-on-chip system, integrating an all-glass microfluidics and on-chip optical detection, was developed and tested. The microfluidic network is etched in a glass substrate, which is then sealed with a glass cover by direct bonding. Thin film amorphous silicon photosensors have been fabricated on the sealed microfluidic substrate preventing the contamination of the micro-channels. The microfluidic network is then made accessible by opening inlets and outlets just prior to the use, ensuring the sterility of the device. The entire fabrication process relies on conventional photolithographic microfabrication techniques and is suitable for low-cost mass production of the device. The lab-on-chip system has been tested by implementing a chemiluminescent biochemical reaction. The inner channel walls of the microfluidic network are chemically functionalized with a layer of polymer brushes and horseradish peroxidase is immobilized into the coated channel. The results demonstrate the successful on-chip detection of hydrogen peroxide down to 18 µM by using luminol and 4-iodophenol as enhancer agent.

The HLA-DR mediated signalling increases the migration and invasion of melanoma cells, the expression and lipid raft recruitment of adhesion receptors, PD-L1 and signal transduction proteins.

The constitutive expression of Major Histocompatibility Complex (MHC) class II molecules is restricted to professional Antigen-Presenting Cells (APCs), nevertheless almost 50% of melanomas express constitutively the MHC class II molecules. Therefore, in two MHC class II constitutive expressing melanoma cell lines we studied the signalling mediated by the HLA-DR molecules in the aim to understand the consequence of class II mediated signalling on metastatic dissemination of melanoma. In particular, we reported that the HLA-DR mediated signalling play a new role in melanoma progression, increasing the migration and invasion of melanoma cells. Furthermore, we showed that the HLA-DR mediated signalling increases the expression and the lipid raft localisation of class II molecules, PD-L1 receptor, Integrin and CAM adhesion receptors, FAK, AKT and STAT3 signalling proteins. We also showed that the HLA-DR mediated signalling increases the activation of FAK, AKT, ERK, PKC and STAT3 signalling proteins and the expression of ILK, PAX, BRAF, ERK and PKC. Indeed, the results showed suggest that the HLA-DR mediated signalling provides a platform useful to frustrate an effective anti-tumour response and to increase melanoma migration and metastatic dissemination of this cancer.

High-dose Ionizing Radiation Regulates Gene Expression Changes in the MCF7 Breast Cancer Cell Line.

BACKGROUND: Intraoperative electron radiation therapy (IOERT) is a therapeutic technique which administers a single high dose of ionizing radiation immediately after surgical tumor removal. IOERT induces a strong stress response: both tumor and normal cells activating pro- and antiproliferative cell signaling pathways. Following treatment, several genes and factors are differently modulated, producing an imbalance in cell fate decision. However, the contribution of these genes and pathways in conferring different cell radiosensitivity and radioresistance needs to be further investigated, in particular after high-dose treatments. Despite the documented and great impact of IOERT in breast cancer care, and the trend for dose escalation, very limited data are available regarding gene-expression profiles and cell networks activated by IOERT or high-dose treatment. The aim of the study was to analyze the main pathways activated following high radiation doses in order to select for potential new biomarkers of radiosensitivity or radioresistance, as well as to identify therapeutic targets useful in cancer care. MATERIALS AND METHODS: We performed gene-expression profiling of the MCF7 human breast carcinoma cell line after treatment with 9- and 23-Gy doses (conventionally used during IOERT boost and exclusive treatments, respectively) by cDNA microarrays. Real-Time Quantitative Reverse Transcription PCR (qRT-PCR), immunofluorescence and immunoblot experiments were performed to validate candidate IOERT biomarkers. We also conducted clonogenic tests and cellular senescence assays to monitor for radiation-induced effects. RESULTS: The analyses highlighted a transcriptome dependent on the dose delivered and a number of specific key genes that may be proposed as new markers of radiosensitivity. Cell and molecular traits observed in MCF7 cells revealed a typical senescent phenotype associated with cell proliferation arrest after treatments with 9- and 23-Gy doses. CONCLUSION: In this study, we report genes and cellular networks activated following high-dose IOERT. The selected validated genes were used to design two descriptive models for each dose delivered. We believe that this study could contribute to the understanding over the complex mechanisms which regulate cell radiosensitivity and radioresistance in order to improve personalized radiotherapeutic treatment.

Glucose level determination with a multi-enzymatic cascade reaction in a functionalized glass chip.

In this work we show the functionalization of the interior of microfluidic glass chips with poly(2-hydroxyethyl methacrylate) polymer brushes as anchors for co-immobilization of the enzymes glucose-oxidase and horseradish peroxidase. The formation of the brush layer and subsequent immobilization of these enzymes have been characterized on flat surfaces by atomic force microscopy and Fourier transform infrared spectroscopy, and studied inside glass chips by field emission scanning microscopy. Enzyme-functionalized glass chips have been applied for performing a multi-enzymatic cascade reaction for the fast (20 s) determination of glucose in human blood samples and the result is in excellent agreement with values obtained from the conventional hospital laboratory. The limit of detection of this bi-enzymatic method is 60 µM. With the advantages of high selectivity and reproducibility, this functionalization method can be used for improving the efficiency of glucose sensors.

Differential occurrence of S100A7 in breast cancer tissues: a proteomic-based investigation.

PURPOSE: The present study reports for the first time a large-scale proteomic screening of the occurrence, subcellular localization and relative quantification of the S100A7 protein among a group of 100 patients, clinically grouped for the diagnosis of infiltrating ductal carcinoma (IDC). EXPERIMENTAL DESIGN: To this purpose, the methods of differential proteomics, Western blotting, and immunohistochemistry were used. RESULTS: The identity of two isoforms of the protein was assessed by mass spectrometry and immunologically confirmed. Moreover, we proved by immunocytochemical applications the exclusive localization of the protein within the neoplastic cells. The correlation of S100A7 expression levels with the collective profile of cancer patients' proteomics predicted functional interactions, distinct for the two isoforms. The S100A7b isoform was significantly correlated with specific protein clusters (calcium binding, signaling and cell motion, heat shock and folding) and intercrossing pathways (antioxidant, metabolic and apoptotic pathways), while the more acidic isoform was correlated with a narrow number of proteins mainly unrelated to the b isoform. CONCLUSIONS AND CLINICAL RELEVANCE: This study is the first proteomic-based report on S100A7 in a large series of IDC patients. The correlation with in silico data may significantly contribute the knowledge of possible pathways for S100A7, providing novel insights into the mechanism of action of this protein. We suggest that each S100A7 isoform is involved in critical phases of the breast cancer growth and progression, probably through interaction with different partner proteins.

Enzyme-functionalized polymer brush films on the inner wall of silicon-glass microreactors with tunable biocatalytic activity.

The lipase from Candida Rugosa was immobilized to a poly(methacrylic acid) polymer brush layer, grown on the inner wall of silicon-glass microreactors. The hydrolysis of 4-nitrophenyl acetate was used as a model reaction to study the activity of this biocatalytic system. The amount of bound lipase could be tuned by changing the polymerization time of the brush formation. The Michaelis-Menten constants and V(max) values, determined for immobilized and free lipase, are similar, demonstrating that the lipase's substrate affinity and its activity remain unchanged upon immobilization to the microchannel wall.

Large-scale proteomic identification of S100 proteins in breast cancer tissues.

BACKGROUND: Attempts to reduce morbidity and mortality in breast cancer is based on efforts to identify novel biomarkers to support prognosis and therapeutic choices. The present study has focussed on S100 proteins as a potentially promising group of markers in cancer development and progression. One reason of interest in this family of proteins is because the majority of the S100 genes are clustered on a region of human chromosome 1q21 that is prone to genomic rearrangements. Moreover, there is increasing evidence that S100 proteins are often up-regulated in many cancers, including breast, and this is frequently associated with tumour progression. METHODS: Samples of breast cancer tissues were obtained during surgical intervention, according to the bioethical recommendations, and cryo-preserved until used. Tissue extracts were submitted to proteomic preparations for 2D-IPG. Protein identification was performed by N-terminal sequencing and/or peptide mass finger printing. RESULTS: The majority of the detected S100 proteins were absent, or present at very low levels, in the non-tumoral tissues adjacent to the primary tumor. This finding strengthens the role of S100 proteins as putative biomarkers. The proteomic screening of 100 cryo-preserved breast cancer tissues showed that some proteins were ubiquitously expressed in almost all patients while others appeared more sporadic. Most, if not all, of the detected S100 members appeared reciprocally correlated. Finally, from the perspective of biomarkers establishment, a promising finding was the observation that patients which developed distant metastases after a three year follow-up showed a general tendency of higher S100 protein expression, compared to the disease-free group. CONCLUSIONS: This article reports for the first time the comparative proteomic screening of several S100 protein members among a large group of breast cancer patients. The results obtained strongly support the hypothesis that a significant deregulation of multiple S100 protein members is associated with breast cancer progression, and suggest that these proteins might act as potential prognostic factors for patient stratification. We propose that this may offer a significant contribution to the knowledge and clinical applications of the S100 protein family to breast cancer.

A brush-gel/metal-nanoparticle hybrid film as an efficient supported catalyst in glass microreactors.

A polymer-brush-based material was applied for the formation and in situ immobilization of silver and palladium nanoparticles, as a catalytic coating on the inner wall of glass microreactors. The brush film was grown directly on the microchannel interior by means of atom-transfer radical polymerization (ATRP), which allows control over the polymer film thickness and therefore permits the tuning of the number of nanoparticles formed on the channel walls. The wide applicability of the catalytic devices is demonstrated for the reduction of 4-nitrophenol and for the Heck reaction.