ISEP – BioMark – Artigos
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- Effect of V concentration in TiSiN monolayer coating on chip formation mechanism and chip sliding velocity during dry turning of Ti–6Al–4V alloyPublication . Kumar, Ch Sateesh; Urbikain, Gorka; Lucio, Pablo Fernández de; Pérez-Salinas, Cristian; Lacalle, Luis Norberto López de; Fernandes, Filipe; Fernandes, FilipeThe current study examines how the self-lubricating characteristics of the novel TiSiVN coating affect the chip formation process and chip sliding velocity during the dry turning of Ti6Al4V titanium alloy. The serration bands tend to straighten at a cutting speed of 125 m/min, which is the main cause of the chips being straightened without tangling for both coated tools. TiSiVN coated tool accounts for higher chip sliding velocity due to the generation of lubricious phases, whereas the higher VS for uncoated tool indicates high tool wear at the highest cutting speed of 125 m/min. Further, r and 0n tend to have an inverse relationship with VS, with 125 m/min cutting speed remaining an exception due to severe changes in tool wear dynamics. The reduction of friction helped to lower the localized strain along the shear bands and the effective stress at the beginning of the formation of the serrated tooth.
- Passive direct methanol fuel cells acting as fully autonomous electrochemical biosensors: Application to sarcosine detectionPublication . Silva Ferreira, Nadia; Carneiro, Liliana P.T; Viezzer, Christian; Almeida, Maria J.T.; Marques, Ana C.; Pinto, Alexandra M.F.R.; Fortunato, Elvira; Ferreira Sales, Maria GoretiThis work describes an innovative electrochemical biosensor that advances its autonomy toward an equipment-free design. The biosensor is powered by a passive direct methanol fuel cell (DMFC) and signals the response via an electrochromic display. Briefly, the anode side of the DMFC power source was modified with a biosensor layer developed using molecularly imprinted polymer (MIP) technology to detect sarcosine (an amino acid derivative that is a potential cancer biomarker). The biosensor layer was anchored on the surface of the anode carbon electrode (carbon black with Pt/Ru, 40:20). This was done by bulk radical polymerization with acrylamide, bis-acrylamide, and vinyl phosphonic acid. This layer selectively interacted with sarcosine when integrated into the passive DMFC (single or multiple, in a stack of 4), which acted as a transducer element in a concentration-dependent process. Serial assembly of a stack of hybrid DMFC/biosensor devices triggered an external electrochromic cell (EC) that produced a colour change. Calibrations showed a concentration-dependent sarcosine response from 3.2 to 2000 µM, which is compatible with the concentration of sarcosine in the blood of prostate cancer patients. The final DMFC/biosensor-EC platform showed a colour change perceptible to the naked eye in the presence of increasing sarcosine concentrations. This colour change was controlled by the DMFC operation, making this approach a self-controlled and self-signalling device. Overall, this approach is a proof-of-concept for a fully autonomous biosensor powered by a chemical fuel. This simple and low-cost approach offers the potential to be deployed anywhere and is particularly suitable for point-of-care (POC) analysis.
- New Quantum-Dot-Based Fluorescent Immunosensor for Cancer Biomarker DetectionPublication . Sousa, Mariana P.; Piloto, Ana Margarida L.; Pereira, Ana Cláudia; Schmitt, Fernando; Fernandes, Ruben; Moreira, Felismina T. C.Cancer antigen 15-3 (CA 15-3) is a biomarker for breast cancer used to monitor response to treatments and disease recurrence. The present work demonstrates the preparation and application of a fluorescent biosensor for ultrasensitive detection of the cancer antigen CA 15-3 protein tumor marker using mercaptopropionic-acid-functionalized cadmium telluride (CdTe@MPA) quantum dots (QDs) conjugated with CA 15-3 antibodies. First, the QDs were synthesized by the hydrothermal route, resulting in spherical nanoparticles up to 3.50 nm in diameter. Subsequently, the QD conjugates were characterized by Fourier transform infrared spectroscopy (FTIR), UV absorption, and fluorescence. The interaction between the conjugates and the protein was studied by fluorescence spectroscopy in buffer and in 10-fold diluted commercial human serum. Calibration in spiked serum samples gave a detection limit of 0.027 U/mL, 1000-fold lower than the clinical limit for CA 15-3 (25 U/mL to 30 U/mL), indicating that this is an ultrasensitive technique. In addition, a rapid response was obtained within 10 min. The biosensor was selective in the presence of the interfering serum proteins BSA, CEA, and CA-125, with a maximum interference of 2% for BSA. The percent recovery was close to 100% with maximum relative standard deviation (RSD%) values of 1.56. Overall, the developed CA 15-3 biosensor provides a simple and sensitive method for ultrasensitive monitoring of breast cancer, as well as the ability to detect other molecules of interest in human serum matrices.
- Dual-Mode Gold Nanoparticle-Based Method for Early Detection of AcanthamoebaPublication . Pastrana, Cristina; Guerreiro, Joana Rafaela; Elumalai, Monisha; Carpena-Torres, Carlos; A, Crooke; Carracedo Rodriguez, Juan Gonzalo; Prado, Marta; Toral, FernandoAcanthamoebakeratitis is an aggressive and rapidly progressing ocular pathology whosemain risk factor is the use of contact lenses. An early and differential diagnosis is considered themain factor to prevent the progression and improve the prognosis of the pathology. However, currentdiagnosis techniques require time, complex and costly materials making an early diagnosis challeng-ing. Thus, there is a need for fast, accessible, and accurate methods forAcanthamoebadetection bypractitioners for timely and suitable treatment and even for contact lens user as preventive diagnosis.Here, we developed a dual-mode colorimetric-based method for fast, visual, and accurate detectionofAcanthamoebausing gold nanoparticles (AuNPs). For this strategy, AuNPs were functionalizedwith thiolated probes and the presence of targetAcanthamoebagenomic sequences, produce a colori-metric change from red to purple. This approach allows the detection of 0.02 and 0.009μM of theunamplifiedAcanthamoebagenome by the naked eye in less than 20 min and by color analysis usinga smartphone. Additionally, real samples were successfully analyzed showing the potential of thetechnology considering the lack of point-of-care tools that are mostly needed.
- Wine astringent compounds monitored by an electrochemical biosensorPublication . Costa, Joana J.; Moreira, Felismina; Soares, Susana; Brandão, Elsa; Mateus, Nuno; Freitas, Victor de; Sales, GoretiAn innovative approach for monitoring astringent polyphenols in beverages (wines) is described, consisting of an electrochemical biosensor constructed by adsorbing salivary α-amylase or proline-rich protein (PRP) onto amined gold screen-printed electrodes. Interaction with polyphenols was tested using pentagalloyl glucose (PGG) as a standard, an important representative element for astringency. The analytical properties of the resulting biosensors were evaluated by electrochemical impedance spectroscopy at different pHs. The PRP-biosensor was able to bind to PGG with higher sensitivity, displaying lower limit of the linear range of 0.6 μM. Wine samples were tested to prove the concept and the concentrations obtained ranged from 0.17 to 4.7 μM, as expressed in PGG units. The effects of side-compounds on PRP and on α-amylase binding to PGG were tested (gallic acid, catechin, ethanol, glucose, fructose and glycerol) and considered negligible. Overall, concentrations > 1.0 μM in PGG units are signaling electrochemical impedance, providing a quantitative monitoring of astringent compounds.
- Molecular Imprinted Polymers on Microneedle Arrays for Point of Care Transdermal Sampling and Sensing of Inflammatory BiomarkersPublication . Oliveira, Daniela; Correia, Barbara P; Sharma, Sanjiv; Moreira, FelisminaThe skin interstitial fluid (ISF) contains biomarkers that complement other biofluids such as blood, sweat, saliva, and urine. It can be sampled in a minimally invasive manner and used either for point of care testing or real time, continuous monitoring of analytes, the latter using microneedle arrays. The analytes present in the skin ISF are indicative of both systemic and local (i.e., skin) physiology. In this paper, we describe combining microneedle technology with molecularly imprinted polymers to demonstrate the potential of transdermal electrochemical sensing. The molecularly imprinted polymer employed here is easy to produce; it can be thought of as plastic antibody. Its synthesis is scalable, and the resulting sensor has a short measurement time (6 min), with high accuracy and a low limit of detection. It provides the requisite specificity to detect the proinflammatory cytokine IL6. IL-6 is present in the skin ISF with other cytokines and is implicated in many clinical states including neurodegenerative diseases and fatal pneumonia from SARSCoV 2. The ability to mass produce microneedle arrays and plastic antibodies will allow for low-cost transdermal sensing devices. The transdermal sensors were able to detect IL-6 at concentrations as low as 1 pg/mL in artificial skin ISF, indicating its utility for routine point of care, bloodless measurements in simpler settings, worldwide.
- Development of colorimetric cellulose-based test-strip for the rapid detection of antibodies against SARS-CoV2 virusPublication . Correia, Bárbara P.; Sousa, Mariana P.; Sousa, Cristina E. A.; Mateus, Daniela; Sebastião, Ana Isabel; Cruz, Maria Teresa; Matos, Ana Miguel; Santos Pereira, Ana Cláudia; Moreira, FelisminaGiven the pandemic situation, there is an urgent need for an accurate test to monitor antibodies anti-SARS-CoV-2, providing crucial epidemiological and clinical information to monitor the evolution of coronavirus disease in 2019 (COVID-19) and to stratify the immunized and asymptomatic population. Therefore, this paper describes a new cellulosebased test strip for rapid and cost-efective quantitative detection of antibodies to SARS-CoV2 virus by colorimetric transduction. For this purpose, Whatman paper was chemically modifed with sodium metaperiodate to introduce aldehyde groups on its surface. Subsequently, the spike protein of the virus is covalently bound by forming an imine group. The chemical control of cellulose paper modifcation was evaluated by Fourier transform infrared spectroscopy, thermogravimetry and contact angle analysis. Colorimetric detection of the antibodies was performed by a conventional staining method using Ponceau S solution as the dye. Color analysis was performed after image acquisition with a smartphone using Image J software. The color intensity varied linearly with the logarithm of the anti-S concentration (from 10 ng/mL to 1 μg/mL) in 500-fold diluted serum samples when plotted against the green coordinate extracted from digital images. The test strip was selective in the presence of nucleocapsid antibodies, urea, glucose, and bovine serum albumin with less than 15% interference, and detection of antibodies in human serum was successfully performed. Overall, this is a simple and afordable design that can be readily used for mass Abstract Given the pandemic situation, there is an population screening and does not require sophisticated equipment or qualifed personnel
- Electrochemical Aptasensor for the Detection of the Key Virulence Factor YadA of Yersinia enterocoliticaPublication . Sande, Maria Georgina; Ferreira, Débora; Rodrigues, Joana; Melo, Luís; Linke, Dirk; Silva, Carla J.; Moreira, Felismina; Sales, Goreti; Rodrigues, LígiaNew point-of-care (POC) diagnosis of bacterial infections are imperative to overcome the deficiencies of conventional methods, such as culture and molecular methods. In this study, we identified new aptamers that bind to the virulence factor Yersinia adhesin A (YadA) of Yersinia enterocolitica using cell-systematic evolution of ligands by exponential enrichment (cell-SELEX). Escherichia coli expressing YadA on the cell surface was used as a target cell. After eight cycles of selection, the final aptamer pool was sequenced by high throughput sequencing using the Illumina Novaseq platform. The sequencing data, analyzed using the Geneious software, was aligned, filtered and demultiplexed to obtain the key nucleotides possibly involved in the target binding. The most promising aptamer candidate, Apt1, bound specifically to YadA with a dissociation constant (Kd) of 11 nM. Apt1 was used to develop a simple electrochemical biosensor with a two-step, label-free design towards the detection of YadA. The sensor surface modifications and its ability to bind successfully and stably to YadA were confirmed by cyclic voltammetry, impedance spectroscopy and square wave voltammetry. The biosensor enabled the detection of YadA in a linear range between 7.0 × 104 and 7.0 × 107 CFU mL−1 and showed a square correlation coefficient >0.99. The standard deviation and the limit of detection was ~2.5% and 7.0 × 104 CFU mL−1, respectively. Overall, the results suggest that this novel biosensor incorporating Apt1 can potentially be used as a sensitive POC detection system to aid the diagnosis of Y. enterocolitica infections. Furthermore, this simple yet innovative approach could be replicated to select aptamers for other (bacterial) targets and to develop the corresponding biosensors for their detection.
- Paper-based ELISA for fast CA 15–3 detection in point-of-carePublication . Carneiro, Mariana C.C.G.; Rodrigues, R. V.; Moreira, Felismina; Sales, Maria Goreti FerreiraThe paper-based Enzyme-Linked Immunosorbent Assay (P-ELISA) is a promising tool for diagnostic applications because the paper matrix is characterised by low cost, short analysis time, portability, and a high surface-to-volume ratio that allows the use of a small sample and reagent volume of a few microliters. In addition, colorimetric assays are suitable for low-resource areas due to their simplicity and naked-eye detectability. Although several works have been reported using paper-based colorimetric sensors for cancer biomarkers, P-ELISA for cancer antigen 15–3 (CA 15–3) has never been reported. Thus, this work reports the development of a rapid, simple and relatively inexpensive paper-based colorimetric assay for the detection of CA 15–3 as cancer biomarker. The assay was developed on a filter paper that was previously washed and chemically oxidized with periodate to generate aldehyde functional groups on the cellulose surface. After covalent binding of the first antibody, detection was performed by a colorimetric reaction based on the oxidation of the substrate 3,3′,5,5′-tetramethylbenzidine (TMB) by a peroxidase enzyme in the presence of H2O2. The colorimetric measurement was based on digital image acquisition analysed using ImageJ software. The linear range of the assay in buffer ranged 2 to 1100 U/mL. The performance of the assay was also successfully tested in human serum from Cormay®, offering a linear range from 2 to 200 U/mL. Thus, this P-ELISA sensor is suitable for the analysis of serum samples, since the physiological value in cancer patients is 30 U/mL. We believe that this proof-of-concept has the potential to be extended to be applied to other protein biomarkers and to be a suitable tool for cancer screening in developing countries.
- Colorimetric Paper-Based Sensors against Cancer BiomarkersPublication . Carneiro, Mariana; Rodrigues, R. V.; Moreira, Felismina; Ferreira Sales, Maria GoretiCancer is a major cause of mortality and morbidity worldwide. Detection and quantification of cancer biomarkers plays a critical role in cancer early diagnosis, screening, and treatment. Clinicians, particularly in developing countries, deal with high costs and limited resources for diagnostic systems. Using low-cost substrates to develop sensor devices could be very helpful. The interest in paper-based sensors with colorimetric detection increased exponentially in the last decade as they meet the criteria for point-of-care (PoC) devices. Cellulose and different nanomaterials have been used as substrate and colorimetric probes, respectively, for these types of devices in their different designs as spot tests, lateral-flow assays, dipsticks, and microfluidic paper-based devices (μPADs), offering low-cost and disposable devices. However, the main challenge with these devices is their low sensitivity and lack of efficiency in performing quantitative measurements. This review includes an overview of the use of paper for the development of sensing devices focusing on colorimetric detection and their application to cancer biomarkers. We highlight recent works reporting the use of paper in the development of colorimetric sensors for cancer biomarkers, such as proteins, nucleic acids, and others. Finally, we discuss the main advantages of these types of devices and highlight their major pitfalls.