Synthesis and Characterization of Cross-Linked Aggregates of Peroxidase from Megathyrsus maximus (Guinea Grass) and Their Application for Indigo Carmine Decolorization.

We present the synthesis of a cross-linking enzyme aggregate (CLEAS) of a peroxidase from Megathyrsus maximus (Guinea Grass) (GGP). The biocatalyst was produced using 50%v/v ethanol and 0.88%w/v glutaraldehyde for 1 h under stirring. The immobilization yield was 93.74% and the specific activity was 36.75 U mg-1. The biocatalyst surpassed by 61% the free enzyme activity at the optimal pH value (pH 6 for both preparations), becoming this increase in activity almost 10-fold at pH 9. GGP-CLEAS exhibited a higher thermal stability (2-4 folds) and was more stable towards hydrogen peroxide than the free enzyme (2-3 folds). GGP-CLEAS removes over 80% of 0.05 mM indigo carmine at pH 5, in the presence of 0.55 mM H2O2 after 60 min of reaction, a much higher value than when using the free enzyme. The operational stability showed a decrease of enzyme activity (over 60% in 4 cycles), very likely related to suicide inhibition.

Commercially available rapid diagnostic tests for the detection of high priority pathogens: status and challenges.

The ongoing COVID-19 pandemic has shown the importance of having analytical devices that allow a simple, fast, and robust detection of pathogens which cause epidemics and pandemics. The information these devices can collect is crucial for health authorities to make effective decisions to contain the disease's advance. The World Health Organization published a list of primary pathogens that have raised concern as potential causes of future pandemics. Unfortunately, there are no rapid diagnostic tests commercially available and approved by the regulatory bodies to detect most of the pathogens listed by the WHO. This report describes these pathogens, the available detection methods, and highlights areas where more attention is needed to produce rapid diagnostic tests for future pandemic surveillance.

Design and characterization of high-affinity synthetic peptides as bioreceptors for diagnosis of cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is one of the illnesses caused by Leishmania parasite infection, which can be asymptomatic or severe according to the infecting Leishmania strain. CL is commonly diagnosed by directly detecting the parasites or their DNA in tissue samples. New diagnostic methodologies target specific proteins (biomarkers) secreted by the parasite during the infection process. However, specific bioreceptors for the in vivo or in vitro detection of these novel biomarkers are rather limited in terms of sensitivity and specificity. For this reason, we here introduce three novel peptides as bioreceptors for the highly sensitive and selective identification of acid phosphatase (sAP) and proteophosphoglycan (PPG), which have a crucial role in leishmaniasis infection. These high-affinity peptides have been designed from the conservative domains of the lectin family, holding the ability to interact with the biological target and produce the same effect than the original protein. The synthetic peptides have been characterized and the affinity and kinetic constants for their interaction with the targets (sAP and PPG) have been determined by a surface plasmon resonance biosensor. Values obtained for KD are in the nanomolar range, which is comparable to high-affinity antibodies, with the additional advantage of a high biochemical stability and simpler production. Pep2854 exhibited a high affinity for sAP (KD = 1.48 nM) while Pep2856 had a good affinity for PPG (KD 1.76 nM). This study evidences that these peptidomimetics represent a novel alternative tool to the use of high molecular weight proteins for biorecognition in the diagnostic test and biosensor devices for CL.

Modulation of the Biocatalytic Properties of a Novel Lipase from Psychrophilic Serratia sp. (USBA-GBX-513) by Different Immobilization Strategies.

In this work, the effect of different immobilization procedures on the properties of a lipase obtained from the extremophilic microorganism Serratia sp. USBA-GBX-513, which was isolated from Paramo soils of Los Nevados National Natural Park (Colombia), is reported. Different Shepharose beads were used: octyl-(OC), octyl-glyoxyl-(OC-GLX), cyanogen bromide (BrCN)-, and Q-Sepharose. The performance of the different immobilized extremophile lipase from Serratia (ESL) was compared with that of the lipase B from Candida antarctica (CALB). In all immobilization tests, hyperactivation of ESL was observed. The highest hyperactivation (10.3) was obtained by immobilization on the OC support. Subsequently, the thermal stability at pH 5, 7, and 9 and the stability in the presence of 50% (v/v) acetonitrile, 50% dioxane, and 50% tetrahydrofuran solvents at pH 7 and 40 °C were evaluated. ESL immobilized on octyl-Sepharose was the most stable biocatalyst at 90 °C and pH 9, while the most stable preparation at pH 5 was ESL immobilized on OC-GLX-Sepharose supports. Finally, in the presence of 50% (v/v) tetrahydrofuran (THF) or dioxane at 40 °C, ESL immobilized on OC-Sepharose was the most stable biocatalyst, while the immobilized preparation of ESL on Q-Sepharose was the most stable one in 40% (v/v) acetonitrile.

Chemoenzymatic Synthesis of the New 3-((2,3-Diacetoxypropanoyl)oxy)propane-1,2-diyl Diacetate Using Immobilized Lipase B from Candida antarctica and Pyridinium Chlorochromate as an Oxidizing Agent.

To exploit the hydrolytic activity and high selectivity of immobilized lipase B from Candida antarctica on octyl agarose (CALB-OC) in the hydrolysis of triacetin and also to produce new value-added compounds from glycerol, this work describes a chemoenzymatic methodology for the synthesis of the new dimeric glycerol ester 3-((2,3-diacetoxypropanoyl)oxy)propane-1,2-diyl diacetate. According to this approach, triacetin was regioselectively hydrolyzed to 1,2-diacetin with CALB-OC. The diglyceride product was subsequently oxidized with pyridinium chlorochromate (PCC) and a dimeric ester was isolated as the only product. It was found that the medium acidity during the PCC treatment and a high 1,2-diacetin concentration favored the formation of the ester. The synthesized compounds were characterized using IR, MS, HR-MS, and NMR techniques. The obtained dimeric ester was evaluated at 100 ppm against seven bacterial strains and two Candida species to identify its antimicrobial activity. The compound has no inhibitory activity against the bacterial strains used but decreased C. albicans and C. parapsilosis growth by 49% and 68%, respectively. Hemolytic activity was evaluated, and the results obtained support the use of the dimeric ester to control C. albicans and C. parapsilosis growth in non-intravenous applications because the compound shows hemolytic activity.

Detection of cancer cells using a peptide nanotube-folic acid modified graphene electrode.

This article describes the preparation of a graphene electrode modified with a new conjugate of peptide nanotubes and folic acid for the selective detection of human cervical cancer cells over-expressing folate receptors. The functionalization of peptide nanotubes with folic acid was confirmed by fluorescence microscopy and atomic force microscopy. The peptide nanotube-folic acid modified graphene electrode was characterized by scanning electron microscopy and cyclic voltammetry. The modification of the graphene electrode with peptide nanotube-folic acid led to an increase in the current signal. The human cervical cancer cells were bound to the modified electrode through the folic acid-folate receptor interaction. Cyclic voltammograms in the presence of [Fe(CN)(6)](3-/4-) as a redox species demonstrated that the binding of the folate receptor from human cervical cancer cells to the peptide nanotube-folic acid modified electrode lowered the electron transfer resulting in a decrease in the measured current. A detection limit of 250 human cervical cancer cells per mL was obtained. Control experiments confirmed that the peptide nanotube-folic acid electrode specifically recognized folate receptors. The modified electrode described here opens up new possibilities for future applications in early stage diagnoses of diseases where cells over-express folate receptors, such as in cancer or leishmaniasis disease.

A new peroxidase from leaves of guinea grass (Panicum maximum): A potential biocatalyst to build amperometric biosensors.

A new plant peroxidase was isolated from the leaves of guinea grass (Panicum maximum) and partially purified using a biphasic polymer system (poly(ethylene glycol) - ammonium sulfate) followed by size-exclusion chromatography and ultracentrifugation until obtaining a homogeneous extract containing a high peroxidase activity. The novel peroxidase was characterized as having a specific activity of 408U/mg and a molecular weight of 30kDa. The pH for its optimum activity was 8.0 and exhibited a high thermostability at 66°C with a kinact of 8.0×10-3min-1. The best substrates for peroxidase from guinea grass are o-dianisidine and 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid). POD from guinea grass was directly immobilized on the surface of a graphene screen printed electrode and cyclic voltammograms in the presence of potassium ferrocyanide ([Fe(CN)6]3-/4-) as a redox species demonstrated an increase in the electron transfer process. The graphene- modified electrode exhibits excellent electrocatalytic activity to the reduction of H2O2, with a linear response in the 100µM to 3.5mM concentration range and a detection limit of 150µM. The new peroxidase from guinea grass allowed the modification of a graphene electrode providing a potential sensor detection system for determination of H2O2 in real samples with some biomedical or environmental importance.

Non-covalent conjugates of single-walled carbon nanotubes and folic acid for interaction with cells over-expressing folate receptors.

We here present a method to form a noncovalent conjugate of single-walled carbon nanotubes and folic acid aimed to interact with cells over-expressing folate receptors. The bonding was obtained without covalent chemical functionalization using a simple, rapid "one pot" synthesis method. The zeta potential for the single-walled carbon nanotube-folic acid solution was -32.4 mV at pH 7.0 and the result indicates that the folic acid coating inhibited aggregation of the carbon nanotubes. Properties of the single-walled carbon nanotube-folic acid conjugate were analyzed using ultraviolet-visible, fluorescence and Raman spectroscopies. While the folic acid fluorescence signature was significantly quenched by the presence of single-walled carbon nanotubes, the Raman spectra of the conjugate displayed a decreased distribution of sp3 sites. Both results were attributed to the noncovalent functionalization of the single-walled carbon nanotubes with folic acid. A more detailed investigation of the single-walled carbon nanotube-folic acid conjugates utilizing scanning electron microscopy, atomic force microscopy and energy-dispersive X-ray spectroscopy confirmed the presence of the well-defined folic acid coating on the individual single-walled carbon nanotubes. The single-walled carbon nanotube-folic acid conjugates were incubated with THP-1 cells and the internalization was evaluated by Giemsa staining with light microscopy, and cytotoxicity was evaluated using the MTT reduction assay. The cytotoxicity studies presented a low toxicity of the conjugates in the THP-1 cells. The low toxicity and the cellular uptake of single-walled carbon nanotube-folic acid by cancer cells suggest their potential use in carbon nanotube-based drug delivery systems and in the diagnosis of cancer or tropical diseases such as leishmaniasis.

Study of the fluorescence quenching of 1-hydroxypyrene-3, 6, 8-trisulfonic acid by single-walled carbon nanotubes / Estudio de la disminución de la fluorescencia del ácido 1-hidroxipyreno-3, 6, 8-trisulfónico por medio de nanotubos de carbono de pared única / Estudo da diminuição da fluorescência do ácido 1-hidroxipireno-3, 6, 8-trisulfônico por meio de nanotubos de carbono de parede simples

Abstract This paper presents a study of the fluorescence quenching of 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS) in the presence of single-walled carbon nanotubes (SWCNT) using a fluorescence method. To investigate the quenching mechanism (dynamic or static) of HPTS, Stern-Volmer plots of single walled carbon nanotubes at different temperatures were used. The positive deviation from linearity in Stern-Volmer plots suggests that single walled carbon nanotubes follow a static quenching mechanism evidenced by the formation of a stable ground state complex. The results presented here help us to clarify the quenching mechanism in the interaction of a pyrene derived dye and carbon nanotubes. This study will open new possibilities in the use of the conjugate formed by SWCNTs and HPTS in the fabrication of a biosensor based on intracellular fluorescent probes.

Resumen Este artículo presenta el estudio de la desactivación de florescencia del ácido 1-hidroxipyreno-3,6,8-trisulfónico (HPTS) en presencia de nanotubos de carbono de pared única (SWCNT). Para investigar el mecanismo de desactivación (dinámico o estático) del HPTS se evaluaron nanotubos de carbono de pared única a diferentes temperaturas y se analizaron por medio de gráficas Stern-Volmer. La desviación positiva de la linealidad en las gráficas Stern-Volmer sugiere que los nanotubos de carbono de pared única actúan por medio de un mecanismo de desactivación estático, que se evidencia también por la formación de un complejo estable en estado fundamental. Los resultados presentados aquí nos ayudan a aclarar el mecanismo de desactivación de fluorescencia cuando tiene lugar la interacción entre un colorante derivado del pireno y nanotubos de carbono. Este estudio abre nuevas posibilidades para el uso de conjugados formados por SWCNT y HPTS en la fabricación de un biosensor basado en sondas fluorescentes intracelulares.

Resumo Este artigo apresentao estudo da diminuicao da fluorescência do ácido 1-hidroxipireno-3,6,8-trisulfônico (HPTS) em presença de nanotubos de carbono de parede simples (SWCNT). Para investigar o mecanismo da desativação (dinâmico ou estático) do HPTS se avaliaram nanotubos de parede simples em diferentes temperaturas e se analisaram por meio de gráficos Stern-Volmer. O desvio positivo de linearidade nos gráficos Stern-Volmer sugere que os nanotubos de carbono de parede simples atuam por meio de um mecanismo de desativação estática, que também se evidencia pela formação de um complexo estável fundamental. Os resultados apresentados aqui nos ajudam a aclarar o mecanismo de desativação de fluorescência quando ocorre a interação entre um corante derivado do pireno e nanotubos de carbono. Este estudo abre novas possibilidades para o uso de conjugados formados por SWCNT e HPTS na fabricação de um biosensor baseado em sondas fluorescentes intracelulares.