Label-free electrochemical immunosensor for Ochratoxin a detection in coffee samples.

Ochratoxin A (OTA) is a nephrotoxic and carcinogenic mycotoxin frequently found in coffee, which directly impacts human health and the economy of many countries. For this reason, there has been a growing need for simple and sensitive tools for the on-site detection of this mycotoxin. In this study, we developed a label-free impedimetric immunosensor to detect OTA. The biosensor was built on a thin-film gold electrode evaporated on glass substrtes, modified with a self-assembled cysteamine monolayer and anti-OTA antibodies. Atomic force microscopy and Microspectroscopy RAMAN confirmed the successful functionalization of the electrodes. The biosensor performance was evaluated by electrochemical impedance spectroscopy and the measurements indicated a linear relationship between the change in the impedance values and the OTA concentration in the range from 0.5 to 100 ng mL-1 with a limit of detection of 0.15 ng mL-1. The biosensor was highly selective and did not suffer matrix interference when analyzed in coffee samples. Furthermore, considering the small sample volumes, the short time required for analysis, and the possibility of miniaturization, the developed biosensor represents a promising analytical device for on-site coffee quality analyses.

Capacitive immunosensor for COVID-19 diagnosis.

COVID-19 has spread worldwide and early detection has been the key to controlling its propagation and preventing severe cases. However, diagnostic devices must be developed using different strategies to avoid a shortage of supplies needed for tests' fabrication caused by their large demand in pandemic situations. Furthermore, some tropical and subtropical countries are also facing epidemics of Dengue and Zika, viruses with similar symptoms in early stages and cross-reactivity in serological tests. Herein, we reported a qualitative immunosensor based on capacitive detection of spike proteins of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19. The sensor device exhibited a good signal-to-noise ratio (SNR) at 1 kHz frequency, with an absolute value of capacitance variation significantly smaller for Dengue and Zika NS1 proteins (|ΔC| = 1.5 ± 1.0 nF and 1.8 ± 1.0 nF, respectively) than for the spike protein (|ΔC| = 7.0 ± 1.8 nF). Under the optimized conditions, the established biosensor is able to indicate that the sample contains target proteins when |ΔC| > 3.8 nF, as determined by the cut-off value (CO). This immunosensor was developed using interdigitated electrodes which require a measurement system with a simple electrical circuit that can be miniaturized to enable point-of-care detection, offering an alternative for COVID-19 diagnosis, especially in areas where there is also a co-incidence of Zika and Dengue.

Electrochemical detection of Zika and Dengue infections using a single chip.

Zika and Dengue are infectious diseases caused by flaviviruses and transmitted by Aedes mosquitoes. Although symptoms are usually mild, complications such as dengue hemorrhagic fever and microcephaly in newborns -after the pregnant woman becomes infected with the Zika virus-have emerged as a global public health concern. The co-circulation of Zika and Dengue viruses and the overlapping of their symptoms represent a challenge for the accurate diagnosis. A single test for the point-of-care detection of both diseases is crucial. Here we report a single chip that distinguishes between Zika and Dengue infections using the non-structural protein 1 (NS1) as biomarkers. A novel multiplex electrochemical device containing four independent working electrodes was developed. Zika and Dengue biosensors were fabricated separately on different working electrodes. Selectivity tests showed that the two biosensors can distinguish not only the NS1 proteins from Zika and Dengue but also the spike proteins present in the SARS-CoV-2. This is especially relevant as patients with COVID-19 may have symptoms similar to Zika and Dengue. The gold surface was modified with cysteamine and antibodies against the NS1 proteins. Both biosensors detected their respective biomarkers at clinically relevant concentrations and presented a good linear relationship between the percentage change in impedance and the logarithm of the NS1 concentration (R2 = 0.990 for Dengue and R2 = 0.995 for Zika). Upon combining a simple sample preparation with a portable detection method, our disposable multiplex device offers a point-of-care diagnostic test for Zika and Dengue using a single chip. Additionally, two other biosensors can be added to the chip, providing a platform for viral detection.

Modulation of beta-amyloid aggregation using ascorbic acid.

Studies have shown that the level of ascorbic acid (AA) is reduced in the brain of Alzheimer's disease (AD) patients. However, its effect on amyloid-ß 1-42 (Aß42) aggregation has not yet been elucidated. Here we investigated for the first time the effect of AA on Aß42 aggregation using fluorescence assay, circular dichroism, atomic force microscopy, isothermal titration calorimetry, ligand docking, and molecular dynamics. Our results showed that the fibril content decreases in the growth phase when the peptides are co-incubated with AA. AA molecules bind to Aß42 peptides with high binding affinity and a binding site for AA between the ß-strands of Aß42 oligomers prevents the stack of adjacent strands. We demonstrate the inhibitory effect of AA on the aggregation of Aß42 and its molecular interactions, which can contribute to the development of an accessible therapy for AD and also to the design of novel drugs for other amyloidogenic diseases.

Exploring silver nanoparticles for cancer therapy and diagnosis.

Nanomaterials have emerged as promising candidates for cancer therapy and diagnosis as they can solve long-term issues such as drug solubility, systemic distribution, tumor acquired resistance, and improve the performance of diagnostic methods. Among inorganic nanomaterials, AgNPs have been extensively studied in the context of cancer treatment and the reported results have raised exciting expectations. In this review, we provide an overview of the recent research on AgNPs antitumoral properties, their application in different cancer treatment modalities, their potential in biosensors development, and also highlight the main challenges and possible strategies to enable its translation to clinical use.

Detecting cancer cells with a highly sensitive LbL-based biosensor.

Early diagnosis of cancer is crucial for therapeutic methods to be more effective and to decrease the mortality rate due to this disease. Current diagnostic methods include imaging techniques that require expensive equipment and specialized personnel, making it difficult to apply them to many patients. To overcome these limitations, many biosensors have been developed to monitor cancer biomarkers. Here, we report on the electrochemical biosensor for selective detection of tumor cells using a simple and low-cost methodology. Layer-by-layer (LbL) self-assembly was used to modify indium tin oxide (ITO) electrodes with alternating layers of polyallylamine hydrochloride (PAH) and folic acid (FA), which binds to overexpressed folate receptors alpha (FRα) in tumor cells. The LbL-based biosensor showed high sensitivity in detecting cervical cancer cells (HeLa cells) using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). A linear dependence with the logarithm cell concentration was observed and excellent detection limits were found, 4 cells mL-1 and 19 cells mL-1 for EIS and CV measurements, respectively. The developed biosensor also presented great reproducibility (RSD = 1.7%) and repeatability (RSD = 1.8%). The selectivity was confirmed after the biosensor interaction with healthy cells (HMEC cells), which did not produce significant changes in the electrochemical signals. Furthermore, it was demonstrated that selective detection of tumor cells occurs via an interaction with FA. The LbL-based biosensor provides a simple, accurate, and cost-effective platform to be applied in the early diagnosis of cancer.

Optimized PAH/Folic acid layer-by-layer films as an electrochemical biosensor for the detection of folate receptors.

Folate receptor alpha (FR-α) is a glycoprotein overexpressed in tumor cell surfaces, especially in gynecologic cancers, and can be used as a biomarker for diagnostics. Currently, FRα is quantified by positron emission tomography (PET) or fluorescence imaging techniques. However, these methods are costly and time-consuming. We report on the development of an electrochemical biosensor for FRα detection based on the use of nanostructured layer-by-layer (LbL) films as modified electrodes. Multilayer films were deposited on indium tin oxide (ITO) electrodes by the alternately assembling of positively charged polyallylamine hydrochloride (PAH) and negatively charged folic acid (FA), used as the biorecognition element. UV-vis and FTIR spectroscopies revealed the successful PAH and FA adsorption on ITO. Devices performance was evaluated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The [PAH/FA] films presented a good reproducibility (RSD of 1.12%) and stability when stored in the Tris-HCl solution (RSD 6.7%). The biosensor electrochemical response exhibited a linear relationship with FRα concentration in the range from 10 to 40 nM. The limit of detection reached for CV and EIS measurements were 0.7 and 1.5 nM, respectively. As a proof-of-concept, we show that the devices can differenciate tumor cells from healthy cell, showing an excellent selectivity. The biosensor device based on [PAH/FA] films represents a promising strategy for a simple, rapid, and low-cost cancer diagnosis through FRα quantification on the surface of cancer cells.

Applications of biosensors in Alzheimer's disease diagnosis.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive and irreversible cognitive decline. Currently, it affects 36 million people and due to population ageing it is estimated that in 2030 disease incidence will reach 60 million individuals. The precise diagnosis of AD is still a complex task, being mainly performed by cerebrospinal fluid (CSF) analysis or neuroimaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI). Despite being effective these techniques are expensive, time-consuming and not accessible for most part of the population. In this scenario biosensors are presented as promising alternatives for simple, rapid and low cost diagnosis of AD. In this revision we summarize the recent advances on biosensors that brings more accessibility to AD diagnosis. We introduce the most used biorecognition elements in miniaturized biosensing systems as well as AD biomarkers present in CSF, in plasma and in genetic material which can be used for disease identification even in early stages. The recent developed biosensors for AD diagnosis using optical, electrochemical and colorimetric techniques as well as their strategies and analytical performances are discussed. Advancements in signal amplification methodologies with nanomaterials to increase biosensors sensitivity are also presented. This review highlights the potential of biosensors to be used as an accurate and portable tool to improve the early AD diagnosis.

Mouse Cytomegalovirus m153 Protein Stabilizes Expression of the Inhibitory NKR-P1B Ligand Clr-b.

Natural killer (NK) cells are a subset of innate lymphoid cells (ILC) capable of recognizing stressed and infected cells through multiple germ line-encoded receptor-ligand interactions. Missing-self recognition involves NK cell sensing of the loss of host-encoded inhibitory ligands on target cells, including MHC class I (MHC-I) molecules and other MHC-I-independent ligands. Mouse cytomegalovirus (MCMV) infection promotes a rapid host-mediated loss of the inhibitory NKR-P1B ligand Clr-b (encoded by Clec2d) on infected cells. Here we provide evidence that an MCMV m145 family member, m153, functions to stabilize cell surface Clr-b during MCMV infection. Ectopic expression of m153 in fibroblasts augments Clr-b cell surface levels. Moreover, infections using m153-deficient MCMV mutants (Δm144-m158 and Δm153) show an accelerated and exacerbated Clr-b downregulation. Importantly, enhanced loss of Clr-b during Δm153 mutant infection reverts to wild-type levels upon exogenous m153 complementation in fibroblasts. While the effects of m153 on Clr-b levels are independent of Clec2d transcription, imaging experiments revealed that the m153 and Clr-b proteins only minimally colocalize within the same subcellular compartments, and tagged versions of the proteins were refractory to coimmunoprecipitation under mild-detergent conditions. Surprisingly, the Δm153 mutant possesses enhanced virulence in vivo, independent of both Clr-b and NKR-P1B, suggesting that m153 potentially targets additional host factors. Nevertheless, the present data highlight a unique mechanism by which MCMV modulates NK ligand expression.IMPORTANCE Cytomegaloviruses are betaherpesviruses that in immunocompromised individuals can lead to severe pathologies. These viruses encode various gene products that serve to evade innate immune recognition. NK cells are among the first immune cells that respond to CMV infection and use germ line-encoded NK cell receptors (NKR) to distinguish healthy from virus-infected cells. One such axis that plays a critical role in NK recognition involves the inhibitory NKR-P1B receptor, which engages the host ligand Clr-b, a molecule commonly lost on stressed cells ("missing-self"). In this study, we discovered that mouse CMV utilizes the m153 glycoprotein to circumvent host-mediated Clr-b downregulation, in order to evade NK recognition. These results highlight a novel MCMV-mediated immune evasion strategy.

Content of Poorly Absorbed Short-Chain Carbohydrates (FODMAP) in Enteral Homemade Diets.

BACKGROUND: Recent studies have shown that commercial enteral formulas with low FODMAP (FO, fermentable oligosaccharides; D, disaccharides; M, monosaccharides; AP, and polyols) content decrease diarrhea of individuals receiving enteral nutrition therapy (ENT). There is no information, however, regarding the content of FODMAP in homemade enteral formula. The objective of this study was to assess energy and macronutrient content, and to quantify the amount of FODMAP content in homemade enteral formula, commonly prescribed for home use after hospital discharge. METHODS: Recipes of homemade enteral formula were requested from all hospitals and healthcare centers in a Brazilian city, totaling 24 recipes. The diets were quantified in relation to their energy content, macronutrients, and FODMAP content. RESULTS: Of the 19 hospitals, 68.4% prescribe only commercial enteral formulas at the time of hospital discharge, and 31.6% prescribe homemade enteral formula, both conventional and commercial. The homemade enteral formula showed a variation from 700 to 3000 kcal/d. The macronutrient levels of carbohydrates, proteins, and fats were 49.0% ± 6.8%, 17.4% ± 3.1%, and 33.5% ± 6.2%, respectively. On average, homemade enteral formula had 58.5% of high FODMAP content, 7.1% of moderate content, and 34.5% of low FODMAP content, considering that milk is the main food responsible for the high content of FODMAP. CONCLUSIONS: Homemade enteral formula presented adequate nutrition characteristics and is rich in FODMAP. It is important to consider FODMAP in enteral diets prescribed for individuals intolerant to these carbohydrates by individualizing the diet prescription.

Metabolic Effects of Cobalt Ferrite Nanoparticles on Cervical Carcinoma Cells and Nontumorigenic Keratinocytes.

The cytotoxic response, cellular uptake, and metabolomic profile of HeLa and HaCaT cell lines treated with cobalt ferrite nanoparticles (CoFe2O4 NPs) were investigated in this study. Cell viability assays showed low cytotoxicity caused by the uptake of the nanoparticles at 2 mg/mL. However, metabolomics revealed that these nanoparticles impacted cell metabolism even when tested at a concentration that presented low cytotoxicity according to the cell viability assay. The two cell lines shared stress-related metabolic changes such as increase in alanine and creatine levels. A reduced level of fumarate was also observed in HeLa cells after treatment with the nanoparticles, and this alteration can inhibit tumorigenesis. Fumarate is considered to be an oncometabolite that can inhibit prolyl hydroxylase, and this inhibition stabilizes HIF1α, one of the master regulators of tumorigenesis that promotes tumor growth and development. In summary, this study showed that nanoparticle-treated HeLa cells demonstrated decreased concentrations of metabolites associated with cell proliferation and tumor growth. The results clearly indicated that treatment with these nanoparticles might cause a perturbation in cellular metabolism.