Clean-Room lithographical processes for the fabrication of Graphene biosensors

This work is on developing clean-room processes for the fabrication of electrolyte-gate graphene field-effect transistors at the wafer scale for biosensing applications. Our fabrication process overcomes two main issues: removing surface residues after graphene patterning and the dielectric passivation of metallic contacts. A graphene residue-free transfer process is achieved by using a pre-transfer, sacrificial metallic mask that protects the entire wafer except the areas around the channel, source, and drain, onto which the graphene film is transferred and later patterned. After the dissolution of the mask, clean gate electrodes are obtained. The multilayer SiO2/SiNx dielectric passivation takes advantage of the excellent adhesion of SiO2 to graphene and the substrate materials and the superior impermeability of SiNx. It hinders native nucleation centers and breaks the propagation of defects through the layers, protecting from prolonged exposition to all common solvents found in biochemistry work, contrary to commonly used polymeric passivation. Since wet etch does not allow the required level of control over the lithographic process, a reactive ion etching process using a sacrificial metallic stopping layer is developed and used for patterning the passivation layer. The process achieves devices with high reproducibility at the wafer scale.Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding UIDB/04650/2020, UIDP/00013/2020, and Operational Program Competitiveness and Internationalization (POCI) under project POCI-01-0145-FEDER-031069 (PORTGRAPHE). This work was partially supported by E.U. Horizon 2020 Research and Innovation Programme, under project MULTIMAL (grant #777222). P.D. Cabral acknowledges the Ph.D. grant (SFRH/BD/128579/2017) from the FC

Attomolar detection of hepatitis C virus core protein powered by molecular antenna-like effect in a graphene field-effect aptasensor

Biosensors based on graphene field-effect transistors have become a promising tool for detecting a broad range of analytes. However, their performance is substantially affected by the functionalization protocol. In this work, we use a controlled in-vacuum physical method for the covalent functionalization of graphene to construct ultrasensitive aptamer-based biosensors (aptasensors) able to detect hepatitis C virus core protein. These devices are highly specific and robust, achieving attomolar detection of the viral protein in human blood plasma. Such an improved sensitivity is rationalized by theoretical calculations showing that induced polarization at the graphene interface, caused by the proximity of covalently bound molecular probe, modulates the charge balance at the graphene/aptamer interface. This charge balance causes a net shift of the Dirac cone providing enhanced sensitivity for the attomolar detection of the target proteins. Such an unexpected effect paves the way for using this kind of graphene-based functionalized platforms for ultrasensitive and real-time diagnostics of different diseases.EU Graphene Flagship funding (Grant Graphene Core3 881603), the Ministerio de Ciencia e Innovación of Spain: PID2020-113142RB-C21, the European Structural Funds via FotoArt-CM project (P2018/NMT-4367) and the Portuguese Foundation for Science and Technology (FCT) via the Strategic Funding UIDB/04650/2020. Work at CAB was funded by the Spanish Ministerio de Ciencia e Innovación (MICINN) grant no. PID2019-104903RB-I00 and the Spanish Agencia Estatal de Investigación (AEI) Project no. MDM-2017-0737 - Unidad de Excelencia “María de Maeztu,” and it also benefits from the interdisciplinary framework provided by CSIC through “LifeHUB.CSIC” initiative (PIE 202120E047-CONEXIONES-LIFE). CIBERehd is funded by Instituto de Salud Carlos III (ISCIII). A.N. is supported by the predoctoral fellowship PRE-CAB-BIOMOLECULAS 2 from INTA. B.T-V. is supported by the predoctoral fellowship TS17/16 from INTA and by the CSIC “Garantía Juvenil” contract CAM19_PRE_CAB_001 funded by Comunidad de Madrid (CAM). FCT supports T.D. and P.C. under Ph.D. grants SFRH/BD/08181/2020 and SFRH/BD/128579/2017. M.M. would like to thank Comunidad de Madrid for the predoctoral grant IND2020/BIO-17523. P.A. and C.B. also acknowledge the support provided by La Caixa Foundation through Project LCF/PR/HR21/52410023. L. V. would like to thank Comunidad de Madrid (TRANSNANOAVANSENS program: S2018-NMT-4349) and E.V. García-Frutos for her assistance during the AFM experiments

Attomolar detection of hepatitis C virus core protein powered by molecular antenna-like effect in a graphene field-effect aptasensor

This study presents the development of a lab-on-a-chip (LoC) by integrating a graphene field-effect transistor (FET) chip with a programmable microfluidic device for DNA detection. The real-time biochemical events on the graphene FET chip were monitored through Dirac voltage shift data from the portable graphene curve reader with changes dependent on the fluidic flow into the sensing interface by a fully automated programmable microfluidic system. High sensitivity with high reliability can be obtained with a nine-graphene sensor layout on a single chip. The portable graphene curve reader also provides a tunable electrical parameter setup and straightforward data acquisition. Fluidic control was performed through a multi-position valve, allowing sequential commands for liquid injection into the polydimethylsiloxane (PDMS) flow cell mounted on the sensing chip. The flow cell design with impinging jet geometry and the microfluidic system packaging offer high precision and portability as a less laborious and low-cost sensing setup. The merged system allows for various functionalities, including probe DNA (pDNA) immobilization, a blocking step, and DNA hybridization with stable signal output autonomously, even in a long-run experimental setup. As a DNA sensor, the proposed prototype has demonstrated a high sensitivity of ~44 mV/decade of target DNA concentration, with an outstanding limit of detection (LoD) of ~0.642 aM, making it one of the most sensitive sensors reported up to date. The programmable device has demonstrated essential versatilities for biomolecular detection in a fully portable and automated platform.This research is supported by PORTGRAPHE-Control of Port and Douro Wines authenticity using graphene DNA sensors project co-funded by Fundação para a Ciência e a Tecnologia (FCT) Portugal (PTDC/BIA-MOL/31069/2017) and the ERDF through COMPETE2020 (POCI-01–0145-FEDER-031069). One of the authors (Telma Domingues) acknowledges a Ph.D. grant from Fundação para a Ciência e a Tecnologia (FCT) Portugal (SFRH/BD/08181/2020). FCT partially supported University of Minho´s research in the Strategic Funding UIDB/04650/2020

Advanced Technologies for Oral Controlled Release: Cyclodextrins for oral controlled release

Cyclodextrins (CDs) are used in oral pharmaceutical formulations, by means of inclusion complexes formation, with the following advantages for the drugs: (1) solubility, dissolution rate, stability and bioavailability enhancement; (2) to modify the drug release site and/or time profile; and (3) to reduce or prevent gastrointestinal side effects and unpleasant smell or taste, to prevent drug-drug or drug-additive interactions, or even to convert oil and liquid drugs into microcrystalline or amorphous powders. A more recent trend focuses on the use of CDs as nanocarriers, a strategy that aims to design versatile delivery systems that can encapsulate drugs with better physicochemical properties for oral delivery. Thus, the aim of this work was to review the applications of the CDs and their hydrophilic derivatives on the solubility enhancement of poorly water soluble drugs in order to increase their dissolution rate and get immediate release, as well as their ability to control (to prolong or to delay) the release of drugs from solid dosage forms, either as complexes with the hydrophilic (e.g. as osmotic pumps) and/ or hydrophobic CDs. New controlled delivery systems based on nanotechonology carriers (nanoparticles and conjugates) have also been reviewed

Loss and Recovery of Mgat3 and GnT-III Mediated E-cadherin N-glycosylation Is a Mechanism Involved in Epithelial-Mesenchymal-Epithelial Transitions

BACKGROUND: N-acetylglucosaminyltransferase-III (GnT-III) is a glycosyltransferase encoded by Mgat3 that catalyzes the addition of β1,4-bisecting-N-acetylglucosamine on N-glycans. GnT-III has been pointed as a metastases suppressor having varying effects on cell adhesion and migration. We have previously described the existence of a functional feedback loop between E-cadherin expression and GnT-III-mediated glycosylation. The effects of GnT-III-mediated glycosylation on E-cadherin expression and cellular phenotype lead us to evaluate Mgat3 and GnT-III-glycosylation role during Epithelial-Mesenchymal-Transition (EMT) and the reverted process, Mesenchymal-Epithelial-Transition (MET). METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the expression profile and genetic mechanism controlling Mgat3 expression as well as GnT-III-mediated glycosylation, in general and specifically on E-cadherin, during EMT/MET. We found that during EMT, Mgat3 expression was dramatically decreased and later recovered when cells returned to an epithelial-like phenotype. We further identified that Mgat3 promoter methylation/demethylation is involved in this expression regulation. The impact of Mgat3 expression variation, along EMT/MET, leads to a variation in the expression levels of the enzymatic product of GnT-III (bisecting GlcNAc structures), and more importantly, to the specific modification of E-cadherin glycosylation with bisecting GlcNAc structures. CONCLUSIONS/SIGNIFICANCE: Altogether, this work identifies for the first time Mgat3 glycogene expression and GnT-III-mediated glycosylation, specifically on E-cadherin, as a novel and major component of the EMT/MET mechanism signature, supporting its role during EMT/MET

Understanding the clinical spectrum of complicated Plasmodium vivax malaria: a systematic review on the contributions of the Brazilian literature

The resurgence of the malaria eradication agenda and the increasing number of severe manifestation reports has contributed to a renewed interested in the Plasmodium vivax infection. It is the most geographically widespread parasite causing human malaria, with around 2.85 billion people living under risk of infection. The Brazilian Amazon region reports more than 50% of the malaria cases in Latin America and since 1990 there is a marked predominance of this species, responsible for 85% of cases in 2009. However, only a few complicated cases of P. vivax have been reported from this region. A systematic review of the Brazilian indexed and non-indexed literature on complicated cases of vivax malaria was performed including published articles, masters' dissertations, doctoral theses and national congresses' abstracts. The following information was retrieved: patient characteristics (demographic, presence of co-morbidities and, whenever possible, associated genetic disorders); description of each major clinical manifestation. As a result, 27 articles, 28 abstracts from scientific events' annals and 13 theses/dissertations were found, only after 1987. Most of the reported information was described in small case series and case reports of patients from all the Amazonian states, and also in travellers from Brazilian non-endemic areas. The more relevant clinical complications were anaemia, thrombocytopaenia, jaundice and acute respiratory distress syndrome, present in all age groups, in addition to other more rare clinical pictures. Complications in pregnant women were also reported. Acute and chronic co-morbidities were frequent, however death was occasional. Clinical atypical cases of malaria are more frequent than published in the indexed literature, probably due to a publication bias. In the Brazilian Amazon (considered to be a low to moderate intensity area of transmission), clinical data are in accordance with the recent findings of severity described in diverse P. vivax endemic areas (especially anaemia in Southeast Asia), however in this region both children and adults are affected. Finally, gaps of knowledge and areas for future research are opportunely pointed out