Paper-based (bio)sensor for label-free detection of 3-nitrotyrosine in human urine samples using molecular imprinted polymer

Over the last years, paper technology has been widely spread as a more affordable, sustainable and reliable support material to be incorporated in the design of point-of-care (POC) diagnostic devices. However, the single work employing a paper-based device for 3-nitrotyrosine (3-NT), a relevant biomarker for oxidative stress (OS) that is a major origin for many diseases, is incapable of reading successfully complex samples because every species that oxidizes before ~0.75?V will also contribute to the final response. Thus, the introduction of a selective element was made into this set-up by including a molecularly-imprinted polymer (MIP) tailored in-situ. Herein, a novel MIP for 3-NT was assembled directly on a paper platform, made conductive with carbon ink and suitable for an electrochemical transduction. The biomimetic material was produced by electropolymerization of phenol after optimizing several experimental parameters, such a scan-rate, number of cycles, range of potential applied, monomer and template concentrations. Under optimal conditions, the label-free sensor was able to respond to 3-NT from 500?nM to 1?mM, yielding a limit of detection of 22.3?nM. Finally, the applicability of the (bio)sensor was tested by performing calibration assays in human urine samples and a good performance was obtained in terms of sensitivity, selectivity and reproducibility. Overall, the attributes of the herein described sensing approach can be compared to a very limited number of other electrochemical devices, that are still using a conventional three electrode system, making this paper-sustained device the first electrochemical (bio)sensor with potential to become a portable and low-cost diagnostic tool for 3-NT. In general, the incorporation of molecular imprinting technology coupled to electrochemical transduction enabled the fabrication of suitable smart sensors for wide screening approaches.Fundação para a Ciência e Tecnologia (FCT) supported this work through FEDER funds from COMPETE 2020 Program and National Funds, under the project PTDC/AAG-TEC/5400/2014 & POCI-01-0145-FEDER-016637, NORTE-01-0145-FEDER-024358, UID/CTM/50025/2019 and the PhD Grants references SFRH/BD/94159/2013 (GVM) and SFRH/BD/115173/2016 (ACM). H2020 also supported this work through the project MindGAP/FET-Open/GA829040.info:eu-repo/semantics/publishedVersio

Paper-based platform with an in situ molecularly imprinted polymer for ß-amyloid

Alzheimers disease (AD) is one of the most common forms of dementia affecting millions of people worldwide. Currently, an easy and effective form of diagnosis is missing, which significantly hinders a possible improvement of the patients quality of life. In this context, biosensors emerge as a future solution, opening the doors for preventive medicine and allowing the premature diagnosis of numerous pathologies. This work presents a pioneering biosensor that combines a bottom-up design approach using paper as a platform for the electrochemical recognition of peptide amyloid -42 (A-42), a biomarker for AD present in blood, associated with visible differences in the brain tissue and responsible for the formation of senile plaques. The sensor layer relies on a molecularly imprinted polymer as a biorecognition element, created on the carbon ink electrodes surface by electropolymerizing a mixture of the target analyte (A-42) and a monomer (O-phenylenediamine) at neutral pH 7.2. Next, the template molecule was removed from the polymeric network by enzymatic and acidic treatments. The vacant sites so obtained preserved the shape of the imprinted protein and were able to rebind the target analyte. Morphological and chemical analyses were performed in order to control the surface modification of the materials. The analytical performance of the biosensor was evaluated by an electroanalytical technique, namely, square wave voltammetry. For this purpose, the analytical response of the biosensor was tested with standard solutions ranging from 0.1 ng/mL to 1 g/mL of A-42. The linear response of the biosensor went down to 0.1 ng/mL. Overall, the developed biosensor offered numerous benefits, such as simplicity, low cost, reproducibility, fast response, and repeatability less than 10%. All together, these features may have a strong impact in the early detection of AD.The authors acknowledge funding from project PTDC/AAGTEC/5400/2014, POCI-01-0145-FEDER-016637, POCI-01-0145-FEDER-007688, and UID/CTM/50025/2019 funded by European funds through FEDER (European Funding or Regional Development) via COMPETE2020 - POCI (operational program for internationalization and competitively) by national funding through the National Foundation for Science and Technology, I.P. (FCT-MCTES). Additionally, they are grateful to the project IBEROS, Instituto de Bioingenieria en Red para el Envejecimiento Saludable, POCTEP/0245-BEROS-1-E, PROGRAMA INTERREG 2014-2020 funded through FEDER within the cooperation region of Galiza/Spain and North of Portugal. A.C.M. and F.T.C.M. gratefully acknowledges FCT-MCTES for the financial support (PhD grant reference SFRH/BD/115173/2016 intituled “Nanobiosensing platform based on MIP-SERS for breast cancer exosome characterization and detection” and Post-Doc grant reference SFRH/BPD/97891/2013 intituled “Biomedical devices for easier and quicker screening procedures of the Alzheimer’s). This work is part of the Master Thesis in Micro and Nanotechnology Engineering defended by Marta V. Pereira. at FCT NOVA titled “Fabrication of 3D electrodes for biosensor applications” in December 2018.info:eu-repo/semantics/publishedVersio

Bacterial cellulose-based microfluidic device for 3D skin modelling (skin-on-chip)

Paper fluidics is based on patterning hydrophilic paper with channels bounded by hydrophobic barriers. Fluids move along channels by capillarity. Several methods are available for patterning paper, with different costs/resolutions. Paper patterning for microfluidics also used the embossing technique to design open-channel microfluidic devices, fabricated by compressing the sheet of paper with the help of 3D plastic printing moulds. These approaches are adopted in this work to develop a multiwell-microplate paper-based microfluidic, aiming the creation of organs-on-chip, combining complexity and miniaturization. Bacterial Cellulose (BC) represents a source of highly pure and biocompatible cellulose, with huge technological potential in many fields - biomedical, composites, textiles, food and cosmetics textiles - but currently still rather underexploited [Gama et al, 2916; Klemm et al, 2018]. This work describes a novel approach towards the development of a nanostructured and multifunctional cellulose-based device for the continuous culture of animal cells and tissues. A multilayered system of modified BC (hydrophobized and electroconductive) was used to assemble the skin-on-chip, a microfluidic platform, using the lab-on-paper technology intended to mimic vascularization, with controlled flow, to introduce external stimuli, such as electrical or mechanical, and to support multicellular growth. This chip serves a multifactorial purpose, aiming the control of each part that make up the overall complex 3D system, including dynamic control of physical, chemical and gaseous gradients, ensure mimetic vascularization, introduce favourable stimuli and co-culture of skin cells. This model sustains cell growth and allow real time and in a high throughput manner to assess cellular phenomena, such as cell-cell crosstalk, paracrine factor exchange, ECM production, as well as tissue homeostasis in the presence of chemical, mechanical, electrical and biological stimuli, and also kinetics of substance delivery on/through the skin. BC hydrophobization was achieved using a new strategy for the surface modification of BC through the combination of oxygen plasma deposition and silanization with trichloromethyl silane. The combined use of the two techniques modifies both the surface roughness and energy and therefore maximizes the hydrophobic effect obtained. These modified membranes were characterized by SEM, water contact angle measurements, FTIR-ATR and XPS, and its cytotoxic potential was investigated using both indirect and direct contact studies with cells. Importantly, this surface modification revealed no short-term cytotoxic effects on L929 and hDNFs cells. This material was used for the construction of a BC-based well plate for cell culture, which can be supplied continuously with culture medium in long term studies (ranging from days to weeks), using a two-layered 3D full-thickness skin equivalent consisting of an epidermal and a dermal tissue layer, cultivated in alginate scaffolds, that can be maintained and studied as a skin surrogate on the SkinChip [Maia et al, 2014].info:eu-repo/semantics/publishedVersio

Water Peel-Off Transfer of Electronically Enhanced, Paper-Based Laser-Induced Graphene for Wearable Electronics

Funding Information: This work is funded by National Funds through FCT I.P., under the scope of the project UIDB/50025/2020-2023. The authors acknowledge the ERC AdG project DIGISMART ref 787410, EC project SYNERGY H2020-WIDESPREAF-2020-5, CSA, proposal number 952169, EC project EMERGE, No. 101008701, and project BEST - ALT20-03-0247-FEDER-113469 | LISBOA-01-0247-FEDER-113469. T.P. and R.C. acknowledge funding from FCT I.P. through the Ph.D. Grants DFA/BD/8606/2020 and UI/BD/151295/2021. The authors also want to thank Jonas Deuermeier for the help with XPS measurements and analysis. Publisher Copyright: © 2022 American Chemical Society.Laser-induced graphene (LIG) has gained preponderance in recent years, as a very attractive material for the fabrication and patterning of graphitic structures and electrodes, for multiple applications in electronics. Typically, polymeric substrates, such as polyimide, have been used as precursor materials, but other organic, more sustainable, and accessible precursor materials have emerged as viable alternatives, including cellulose substrates. However, these substrates have lacked the conductive and chemical properties achieved by conventional LIG precursor substrates and have not been translated into fully flexible, wearable scenarios. In this work, we expand the conductive properties of paper-based LIG, by boosting the graphitization potential of paper, through the introduction of external aromatic moieties and meticulous control of laser fluence. Colored wax printing over the paper substrates introduces aromatic chemical structures, allowing for the synthesis of LIG chemical structures with sheet resistances as low as 5 ω·sq-1, translating to an apparent conductivity as high as 28.2 S·cm-1. Regarding chemical properties, ID/IG ratios of 0.28 showcase low defect densities of LIG chemical structures and improve on previous reports on paper-based LIG, where sheet resistance has been limited to values around 30 ω·sq-1, with more defect dense and less crystalline chemical structures. With these improved properties, a simple transfer methodology was developed, based on a water-induced peel-off process that efficiently separates patterned LIG structures from the native paper substrates to conformable, flexible substrates, harnessing the multifunctional capabilities of LIG toward multiple applications in wearable electronics. Proof-of concept electrodes for electrochemical sensors, strain sensors, and in-plane microsupercapacitors were patterned, transferred, and characterized, using paper as a high-value LIG precursor for multiples scenarios in wearable technologies, for improved sustainability and accessibility of such applications.publishersversionpublishe

Paper-based laser-induced graphene for sustainable and flexible microsupercapacitor applications

Funding Information: Open access funding provided by FCT|FCCN (b-on). This work was financed by national funds from Fundação para a Ciência e a Tecnologia (FCT), I.P., in the scope of the projects LA/P/0037/2020, UIDP/50025/2020, and UIDB/50025/2020 of the Associate Laboratory Institute of Nanostructures, Nanomodelling and Nanofabrication–i3N and by FEDER funds through the COMPETE 2020 Program and National Funds through Portuguese Foundation for Science and Technology under projects POCI-01–0145-FEDER-007688, UID/CTM/50025 and by ERC AdG grant from the project DIGISMART (ERC-AdG-2017, GA 787410). J.C. would like to acknowledge FCT/MCTES for his present research contract with reference CEECIND/00880/2018. R.C. acknowledges funding from i3N-FCT I.P. through the PhD Grant UI/BD/151295/2021. S. S. and T. P. also acknowledge the funding from National Foundation for Science and Technology, through the PhD Grants SFRH/BD/149751/2019 and 2020.08606.BD, respectively. Publisher Copyright: © 2022, The Author(s).Laser-induced graphene (LIG) is as a promising material for flexible microsupercapacitors (MSCs) due to its simple and cost-effective processing. However, LIG-MSC research and production has been centered on non-sustainable polymeric substrates, such as polyimide. In this work, it is presented a cost-effective, reproducible, and robust approach for the preparation of LIG structures via a one-step laser direct writing on chromatography paper. The developed strategy relies on soaking the paper in a 0.1 M sodium tetraborate solution (borax) prior to the laser processing. Borax acts as a fire-retardant agent, thus allowing the laser processing of sensitive substrates that other way would be easily destroyed under the high-energy beam. LIG on paper exhibiting low sheet resistance (30 Ω sq−1) and improved electrode/electrolyte interface was obtained by the proposed method. When used as microsupercapacitor electrodes, this laser-induced graphene resulted in specific capacitances of 4.6 mF cm−2 (0.015 mA cm−2). Furthermore, the devices exhibit excellent cycling stability (> 10,000 cycles at 0.5 mA cm−2) and good mechanical properties. By connecting the devices in series and parallel, it was also possible to control the voltage and energy delivered by the system. Thus, paper-based LIG-MSC can be used as energy storage devices for flexible, low-cost, and portable electronics. Additionally, due to their flexible design and architecture, they can be easily adapted to other circuits and applications with different power requirements. Graphical Abstract: [Figure not available: see fulltext.]publishersversionpublishe

Paper-based in-situ gold nanoparticle synthesis for colorimetric, non-enzymatic glucose level determination

ref. 787410 SFRH/BD/115173/2016 SFRH/BD/132057/2017Due to its properties, paper represents an alternative to perform point-of-care tests for colorimetric determination of glucose levels, providing simple, rapid, and inexpensive means of diagnosis. In this work, we report the development of a novel, rapid, disposable, inexpensive, enzyme-free, and colorimetric paper-based assay for glucose level determination. This sensing strategy is based on the synthesis of gold nanoparticles (AuNPs) by reduction of a gold salt precursor, in which glucose acts simultaneously as reducing and capping agent. This leads to a direct measurement of glucose without any enzymes or depending on the detection of intermediate products as in conventional enzymatic colorimetric methods. Firstly, we modelled the synthesis reaction of AuNPs to determine the optical, morphological, and kinetic properties and their manipulation for glucose sensing, by determining the influence of each of the reaction precursors towards the produced AuNPs, providing a guide for the manipulation of nucleation and growth. The adaptation of this synthesis into the developed paper platform was tested and calibrated using different standard solutions with physiological concentrations of glucose. The response of the colorimetric signals obtained with this paper-based platform showed a linear behavior until 20 mM, required for glycemic control in diabetes, using the Red × Value/Grey feature combination as a calibration metric, to describe the variations in color intensity and hue in the spot test zone. The colorimetric sensor revealed a detection limit of 0.65 mM, depending on calibration metric and sensitivity of 0.013 AU/mM for a linear sensitivity range from 1.25 to 20 mM, with high specificity for the determination of glucose in complex standards with other common reducing interferents and human serum.publishersversionpublishe

Surface modification of starch based blends using potassium permanganate-nitric acid system and its effect on the adherence and proliferation of osteoblastic-like cells

The surface modification of three starch based polymeric biomaterials, using a KMnO4/NHO3 oxidizing system, and the effect of that modification on the osteoblastic cell adhesion has been investigated. The rationale of this work is as follows—starch based polymers have been proposed for use as tissue engineering scaffolds in several publications. It is known that in biodegradable systems it is quite difficult to have both cell adhesion and proliferation. Starch based polymers have shown to perform better than poly-lactic acid based materials but there is still room for improvement. This particular work is aimed at enhancing cell adhesion and proliferation on the surface of several starch based polymer blends that are being proposed as tissue engineering scaffolds. The surface of the polymeric biomaterials was chemically modified using a KMnO4/HNO3 system. This treatment resulted in more hydrophilic surfaces, which was confirmed by contact angle measurements. The effect of the treatment on the bioactivity of the surface modified biomaterials was also studied. The bioactivity tests, performed in simulated body fluid after biomimetic coating, showed that a dense film of calcium phosphate was formed after 30 days. Finally, human osteoblast-like cells were cultured on unmodified (control) and modified materials in order to observe the effect of the presence of higher numbers of polar groups on the adhesion and proliferation of those cells. Two of the modified polymers presented changes in the adhesion behavior and a significant increase in the proliferation rate kinetics when compared to the unmodified controls.FCT (Portugal) for providing the postdoctoral grant (BPD/8491/2002)

Surface modification of starch based biomaterials by oxygen plasma or UV-irradiation

Radiation is widely used in biomaterials science for surface modification and sterilization. Herein, we describe the use of plasma and UV-irradiation to improve the biocompatibility of different starch-based blends in terms of cell adhesion and proliferation. Physical and chemical changes, introduced by the used methods, were evaluated by complementary techniques for surface analysis such as scanning electron microscopy, atomic force microscopy, contact angle analysis and X-ray photoelectron spectroscopy. The effect of the changed surface properties on the adhesion of osteoblast-like cells was studied by a direct contact assay. Generally, both treatments resulted in higher number of cells adhered to the modified surfaces. The importance of the improved biocompatibility resulting from the irradiation methods is further supported by the knowledge that both UV and plasma treatments can be used as cost-effective methods for sterilization of biomedical materials and devices.I. P. thanks the FCT for providing her a postdoctoral scholarship (SFRH/BPD/8491/2002). This work was partially supported by FCT, through funds from the POCTI and/or FEDER programs, The European Union funded STREP Project HIPPOCRATES (NNM-3-CT-2003-505758) and the European NoE EXPERTISSUES (NMP3-CT-2004-500283)

Worsening of Cardiomyopathy Using Deflazacort in an Animal Model Rescued by Gene Therapy

We have previously demonstrated that gene therapy can rescue the phenotype and extend lifespan in the delta-sarcoglycan deficient cardiomyopathic hamster. In patients with similar genetic defects, steroids have been largely used to slow down disease progression. Aim of our study was to evaluate the combined effects of steroid treatment and gene therapy on cardiac function. We injected the human delta-sarcoglycan cDNA by adeno-associated virus (AAV) 2/8 by a single intraperitoneal injection into BIO14.6 Syrian hamsters at ten days of age to rescue the phenotype. We then treated the hamsters with deflazacort. Treatment was administered to half of the hamsters that had received the AAV and the other hamsters without AAV, as well as to normal hamsters. Both horizontal and vertical activities were greatly enhanced by deflazacort in all groups. As in previous experiments, the AAV treatment alone was able to preserve the ejection fraction (70±7% EF). However, the EF value declined (52±14%) with a combination of AAV and deflazacort. This was similar with all the other groups of affected animals. We confirm that gene therapy improves cardiac function in the BIO14.6 hamsters. Our results suggest that deflazacort is ineffective and may also have a negative impact on the cardiomyopathy rescue, possibly by boosting motor activity. This is unexpected and may have significance in terms of the lifestyle recommendations for patients