Rapid and selective detection of dopamine in human serum using an electrochemical sensor based on zinc oxide nanoparticles, nickel phthalocyanines, and carbon nanotubes.

Composite materials have gained significant attention owing to the synergistic effects of their constituent materials, thereby facilitating their utilization in new applications or in improving the existing ones. In this study, a composite based on nickel phthalocyanine (NiTsPc), zinc oxide nanoparticles (ZnONPs), and carbon nanotubes (CNT) was developed and subsequently immobilized on a pyrolytic graphite electrode (PGE). The PGE/NiTsPc-ZnONPs-CNT was identified as a selective catalytic hybrid system for detection of neurotransmitter dopamine (DA). The electrochemical and morphological characterizations were conducted using atomic force microscopy (AFM). Chronoamperometry and differential pulse voltammetry (DPV) were used to detect DA and detection limits of 24 nM and 7.0 nM was found, respectively. In addition, the effects of some possible DA interferents, such as ascorbic acid, uric acid, and serotonin, on DA response were evaluated. Their presence did not show significant variations in the DA electrochemical response. The high specificity and sensitivity of PGE/NiTsPc-ZnONPs-CNT for DA enabled its direct detection in human serum without sample pretreatment as well as in DA-enriched serum samples, whose recovery levels were close to 100%, thereby confirming the effectiveness of the proposed method. In general, PGE/NiTsPc-ZnONPs-CNT is a promising candidate for future applications in clinical diagnosis.

Poly(Alizarin Red S) on pyrolytic graphite electrodes as a new multi-electronic system for sensing oxandrolone in urine.

This study presents a new polymeric and multielectronic system, the poly-Alizarin Red S (PARS), obtained from the electropolymerization of Alizarin Red S (ARS) dye on an edge-plane pyrolytic graphite electrode (EPPGE) surface. During EPPGE/PARS electrochemical characterization, we identified seven stable and reversible redox peaks in acidic medium (0.10 mol L-1, pH 1.62 KH2PO4), which indicated its mechanisms underlying electropolymerization and electrochemical behavior. To the best of our knowledge, this is the first study to use an EPPGE/PARS electrode to detect oxandrolone (OXA) in artificial urine, where PARS acts as a synthetic receptor for OXA. The interactions of OXA with EPPGE/PARS as well as the properties of PARS were investigated using density functional theory (DFT). Atomic force microscopy (AFM) was used to characterize EPPGE/PARS, and it was found that the PARS polymer formed a semi-globular phase on the EPPGE surface. The limit of detection for OXA found by the sensor was close to 0.50 nmol L-1, with a recovery rate of approximately 100% in artificial urine. In addition to the application proposed in this study, EPPGE/PARS is a low-cost product that could be applied in several devices and processes, such as supercapacitors and electrocatalysis.

Cashew-gum-based silver nanoparticles and palygorskite as green nanocomposites for antibacterial applications.

Nanocomposite materials have been proposed to enhance the properties of different materials. In this study, palygorskite (Pal) clay is proposed as a support matrix for silver nanoparticles stabilised with cashew gum (Anacardium occidentale L.) (AgNPs-CG), producing the Pal/AgNPs-CG nanocomposite, whose bactericidal activity was studied. AgNPs-CG was synthesised using a green method in which CG acted as a reducing and stabilising agent for these nanostructures. AgNPs-CGs were subsequently characterised then adsorbed to the Pal surface, which was previously treated to remove impurities such as quartz. Pal and Pal/AgNPs-CG were characterised by X-ray diffraction, specific surface area, thermal analysis, Fourier transform infrared spectroscopy, scanning electron microscopy, energy dispersive spectroscopy, and transmission electron microscopy. The antibacterial activity assay by the direct contact method showed that the synergistic effect of the combination of AgNPs-CG and Pal increased the bactericidal effect of the nanomaterial compared with the AgNPs-CG activity, reaching a percentage inhibition of up to 70.2% against E. coli and 85.3% against S. aureus. Nanocomposite atoxicity was demonstrated by the Artemia Salina model. Thus, the Pal/AgNPs-CG nanocomposite emerges as a nanomaterial with potential antibacterial applications.

Development of cashew gum-based bionanocomposite as a platform for electrochemical trials.

Cashew gum (CG) biopolymer is from natural source, biocompatible, non-toxic, inexpensive, and easily extracted from the exudate of the Anacardium occidentale L. tree, which is abundant in the north and northeast of Brazil. Prussian blue nanoparticles (PBNPs) can be embedded in the natural materials and have been used in several (bio)technological applications. This work presents a new cashew gum-based bionanocomposite containing PBNPs prepared in situ (PBNPs@GC), where the CG biopolymer was used as a matrix to prevent nanoparticles aggregation. Herein, investigate the effect of different CG concentrations about the bionanocomposite properties, and demonstrated it use as potential electrochemical sensor for drugs trials. The PBNPs@CG were characterized using UV-Vis, FTIR, XRD, DLS, zeta potential, TEM and cyclic voltammetry. The CG proved to be a suitable host for the PBNPs synthetized. These PBNPs were spherical, crystalline, stable, with a size of 5.0-15.0 nm, and without agglomeration. The bionanocomposite electrochemical behavior shown their ability to the oxidize some drugs, such as metamizole (MTM), acetaminophen (ACT) and methotrexate (MTX). These results demonstrated the innovative character of these bionanocomposite and encourage their further exploration for applications in nanobiomedicine like electrochemical (bio)sensors.

Synthesis of catalyst composed of palygorskita-TiO2 and silver nanoparticles for the development of assays antioxidant based on the generation of reactive oxygen species.

The great interest in compounds that present antioxidant capacity has generating the urgent need for analytical methods that could determine the antioxidant potential of these sources. A method based on generation of reactive oxygen species in water from catalyst composed of palygorskita-TiO2 and silver nanoparticles (AgNPs/TiO2-PAL) was developed and applied to antioxidant assays. Silver nanoparticles were synthesized using silver nitrate solution, sodium borohydride reducing agent and Caraia gum as stabilizing agent. Incorporation of AgNPs into the previously synthesized TiO2-PAL was performed. The catalyst AgNPs/TiO2-PAL was characterized by UV-vis spectroscopy, X-ray diffractometry and scanning electron microscopy. The catalyst AgNPs/TiO2-PAL were used to perform an antioxidant activity method which consisted in monitoring the discoloration of acid yellow 73 dye (AY73) in the presence of gallic acid antioxidant comparing to the dye discoloration in the absence of the antioxidant. A microplate reader was used to measure the discoloration of the aqueous solutions of AY73, irradiated by UV light for 60 min. The effect of reactive oxygen species generated by AgNPs/TiO2-PAL based in photocatalytic kinetics of AY73 dye was investigated. The oxidation of AY73 dye by photocatalysis in the system with AgNPs/TiO2-PAL catalysts was carried out mainly by the participation of O2 ·-, HO· and 1O2 species, in this order of importance. The results showed that the synthesis of the AgNPs/TiO2-PAL catalyst was successfully carried out and the application of this material in the development of an innovative methodology for the determination of antioxidant activity was extremely promising.

Development of a low-cost electrochemical sensor based on babassu mesocarp (Orbignya phalerata) immobilized on a flexible gold electrode for applications in sensors for 5-fluorouracil chemotherapeutics.

There are increasing concerns regarding the risks arising from the contamination of manipulators of antineoplastic drugs promoted by occupational exposure or even in the dosage of drugs. The present work proposes the use of an electrochemical sensor based on a biopolymer extracted from the babassu coconut (Orbignya phalerata) for the determination of an antineoplastic 5-fluorouracil (5-FU) drug as an alternative for the monitoring of these drugs. In order to reduce the cost of this sensor, a flexible gold electrode (FEAu) is proposed. The surface modification of FEAu was performed with the deposition of a casting film of the biopolymer extracted from the babassu mesocarp (BM) and modified with phthalic anhydride (BMPA). The electrochemical activity of the modified electrode was characterized by cyclic voltammetry (CV), and its morphology was observed by atomic force microscopy (AFM). The FEAu/BMPA showed a high sensitivity (8.8 µA/µmol/L) and low limit of detection (0.34 µmol/L) for the 5-FU drug in an acid medium. Electrochemical sensors developed from the babassu mesocarp may be a viable alternative for the monitoring of the 5-FU antineoplastic in pharmaceutical formulations, because in addition to being sensitive to this drug, they are constructed of a natural polymer, renewable, and abundant in nature. Graphical abstract ᅟ.

Lateral Flow Assay for Interleukin 6: A Technological and Scientific Prospection of a 10-Year Survey.

BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that plays a crucial role not only in the immune system but also in numerous biological processes. Elevated IL-6 levels have been observed in many types of cancer and inflammatory diseases. Among the techniques that have been used to quantify IL-6, a Lateral Flow Assay (LFA) is one of the most promising. OBJECTIVE: The objective of this study was to perform a technological and scientific exploration of the development of an LFA for IL-6. METHOD: The Instituto Nacional de Propriedade Industrial-INPI (Brazil), European Patent Office - Espacenet, and United States Patent and Trademark Office-USPTO were used for the technological prospection to consider all the patents regarding an LFA for IL-6 detection. For the scientific investigation, the following periodic databases were used: Web of Science, Science Direct, PubMed, and Scielo, and were monitored from 2007 to May 2017. RESULTS: We found no patents based on the searched keywords, and a limited number of scientific articles were found. Therefore, the development of an LFA sensor for the detection of IL-6 appears to be innovative with significant biotechnological importance, creating a technology transfer to promote economic and industrial growth. CONCLUSION: The development of lateral flow immunosensors for IL-6 is innovative, as we could not find patents describing such a biosensor. Scientific prospection reported two lateral flow immunosensors. However, the test for IL-6 cannot be completed without using some kind of equipment for detection, such as the one we propose in future developments.

Electrochemical sensors and biosensors for the analysis of antineoplastic drugs.

Cancer is a leading cause of death worldwide, often being treated with antineoplastic drugs that have high potential for toxicity to humans and the environment, even at very low concentrations. Therefore, monitoring these drugs is of utmost importance. Among the techniques used to detect substances at low concentrations, electrochemical sensors and biosensors have been noted for their practicality and low cost. This review brings, for the first time, a simplified outline of the main electrochemical sensors and biosensors developed for the analysis of antineoplastic drugs. The drugs analyzed and the methodology used for electrochemical sensing are described, as are the techniques used for drug quantification and the analytical performance of each sensor, highlighting the limit of detection (LOD), as well as the linear range of quantification (LR) for each system. Finally, we present a technological prospection on the development and use of electrochemical sensors and biosensors in the quantification of antineoplastic drugs. A search of international patent databases revealed no patents currently submitted under this topic, suggesting this is an area to be further explored. We also show that the use of these systems has been gaining prominence in recent years, and that the quantification of antineoplastic drugs using electrochemical techniques could bring great financial and health benefits.

Layer-by-layer films containing peptides of the Cry1Ab16 toxin from Bacillus thuringiensis for potential biotechnological applications.

Cry1Ab16 is a toxin of crystalline insecticidal proteins that has been widely used in genetically modified organisms (GMOs) to gain resistance to pests. For the first time, in this study, peptides derived from the immunogenic Cry1Ab16 toxin (from Bacillus thuringiensis) were immobilized as layer-by-layer (LbL) films. Given the concern about food and environmental safety, a peptide with immunogenic potential, PcL342-354C, was selected for characterization of the electrochemical, optical, and morphological properties. The results obtained by cyclic voltammetry (CV) showed that the peptide have an irreversible oxidation process in electrolyte of 0.1 mol · L(-1) potassium phosphate buffer (PBS) at pH7.2. It was also observed that the electrochemical response of the peptide is governed mainly by charge transfer. In an attempt to maximize the electrochemical signal of peptide, it was intercalated with natural (agar, alginate and chitosan) or synthetic polymers (polyethylenimine (PEI) and poly(sodium 4-styrenesulfonate (PSS)). The presence of synthetic polymers on the film increased the electrochemical signal of PcL342-354C up to 100 times. Images by Atomic Force Microscopy (AFM) showed that the immobilized PcL342-354C formed self-assembled nanofibers with diameters ranging from 100 to 200 nm on the polymeric film. By UV-Visible spectroscopy (UV-Vis) it was observed that the ITO/PEI/PSS/PcL342-354C film grows linearly up to the fifth layer, thereafter tending to saturation. X-ray diffraction confirmed the presence on the films of crystalline ITO and amorphous polypeptide phases. In general, the ITO/PEI/PSS/PcL342-354C film characterization proved that this system is an excellent candidate for applications in electrochemical sensors and other biotechnological applications for GMOs and environmental indicators.

Immobilization of cationic antimicrobial peptides and natural cashew gum in nanosheet systems for the investigation of anti-leishmanial activity.

This report details the development of thin films containing an antimicrobial peptide, specifically, dermaseptin 01 (GLWSTIKQKGKEAAIAAA-KAAGQAALGAL-NH2, [DRS 01]), and a natural polysaccharide, for a novel application in detecting the presence of Leishmania cells and maintaining anti-leishmanial activity. The peptide DRS 01 was immobilized in conjunction with natural cashew gum (CG) onto an indium tin oxide (ITO) substrate using the Layer-by-Layer (LbL) deposition technique. The LbL film ITO/CG/DRS 01, containing DRS 01 as the outer layer, was capable of detecting the presence of Leishmania cells and acting as an anti-leishmanial system. Detection was performed using cyclic voltammetry (CV) in phosphate buffer (pH7.2) in the presence of promastigote cells (0-10(7)cells/mL). The results showed a linear and inversely proportional relation between the concentration of Leishmania infantum protozoan cells and the measured current values obtained for the films, which was attributed to the effect of peptide-induced lysis of the cell membrane, and resulted in freed residues that were adsorbed on the electrode surface. With this, the paper shows a method using thin films with this new material to demonstrate the anti-leishmanial activity in vitro models of carpet-like mechanisms.

Nanostructured layer-by-layer films containing phaeophytin-b: electrochemical characterization for sensing purposes.

This paper reports the study and characterization of a new platform for practical applications, where the use of phaeophytin-b (phaeo-b), a compound derived from chlorophyll, was characterized and investigated for sensing purposes. Modified electrodes with nanostructured phaeo-b films were fabricated via the layer-by-layer (LbL) technique, where phaeo-b was assembled with cashew gum, a polysaccharide, or with poly(allylamine) hydrochloride (PAH). The multilayer formation was investigated with UV-Vis spectroscopy by monitoring the absorption band associated to phaeo-b at approximately 410 nm, where distinct molecular interactions between the materials were verified. The morphology of the films was analyzed by atomic force microscopy (AFM). The electrochemical properties through redox behavior of phaeo-b were studied with cyclic voltammetry. The produced films were applied as sensors for hydrogen peroxide (H2O2) detection. In terms of sensing, the cashew/phaeo-b film exhibited the most promising result, with a fast response and broad linear range upon the addition of H2O2. This approach provides a simple and inexpensive method for development of a nonenzymatic electrochemical sensor for H2O2.

Collagen-based silver nanoparticles for biological applications: synthesis and characterization.

BACKGROUND: Type I collagen is an abundant natural polymer with several applications in medicine as matrix to regenerate tissues. Silver nanoparticles is an important nanotechnology material with many utilities in some areas such as medicine, biology and chemistry. The present study focused on the synthesis of silver nanoparticles (AgNPs) stabilized with type I collagen (AgNPcol) to build a nanomaterial with biological utility. Three formulations of AgNPcol were physicochemical characterized, antibacterial activity in vitro and cell viability assays were analyzed. AgNPcol was characterized by means of the following: ultraviolet-visible spectroscopy, dynamic light scattering analysis, Fourier transform infrared spectroscopy, atomic absorption analysis, transmission electron microscopy and of X-ray diffraction analysis. RESULTS: All AgNPcol showed spherical and positive zeta potential. The AgNPcol at a molar ratio of 1:6 showed better characteristics, smaller hydrodynamic diameter (64.34 ± 16.05) and polydispersity index (0.40 ± 0.05), and higher absorbance and silver reduction efficiency (0.645 mM), when compared with the particles prepared in other mixing ratios. Furthermore, these particles showed antimicrobial activity against both Staphylococcus aureus and Escherichia coli and no toxicity to the cells at the examined concentrations. CONCLUSIONS: The resulted particles exhibited favorable characteristics, including the spherical shape, diameter between 64.34 nm and 81.76 nm, positive zeta potential, antibacterial activity, and non-toxicity to the tested cells (OSCC).

Development and characterization of multilayer films of polyaniline, titanium dioxide and CTAB for potential antimicrobial applications.

Composites prepared from polyaniline (PANI) and the ceramic technology of titanium dioxide (TiO2) have been proposed, however, the interaction of these materials with greater control of molecular arrangement becomes attractive in order to achieve properties not previously described or yet the optimization of those already reported. Therefore, in this study, thin hybrid films made of polyaniline (PANI), a conductive polymer, and the technological ceramic, titanium dioxide (TiO2), were prepared by the layer-by-layer (LbL) self-assembly technique. The films were characterized by cyclic voltammetry (CV), UV-VIS spectroscopy and atomic force microscopy (AFM). Aiming to improve the dispersion of the ceramic in the polymer matrix, the commercial surfactant, cetyl trimethylammonium bromide (CTAB), was used in the formation of the films. The best condition of deposition was found showing synergic interactions between the conjugated materials. The antibacterial activity of the PANI(TiO2)/CTAB films was studied and the obtained results suggest their use as antimicrobial coatings.

Development and antibacterial activity of cashew gum-based silver nanoparticles.

The present study describes the development of a green synthesis of silver nanoparticles reduced and stabilized by exuded gum from Anacardium occidentale L. and evaluates in vitro their antibacterial and cytotoxic activities. Characterization of cashew gum-based silver nanoparticles (AgNPs) was carried out based on UV-Vis spectroscopy, transmission electron microscopy and dynamic light scattering analysis which revealed that the synthesized silver nanoparticles were spherical in shape, measuring about 4 nm in size with a uniform dispersal. AgNPs presented antibacterial activity, especially against Gram-negative bacteria, in concentrations where no significant cytotoxicity was observed.

Multilayer Films Electrodes Consisted of Cashew Gum and Polyaniline Assembled by the Layer-by-Layer Technique: Electrochemical Characterization and Its Use for Dopamine Determination.

We take advantage of polyelectrolyte feature exhibited by natural cashew gum (Anacardium occidentale L.) (CG), found in northeast Brazil, to employ it in the formation of electroactive nanocomposites prepared by layer-by-layer (LbL) technique. We used polyaniline unmodified (PANI) or modified with phosphonic acid (PA), PANI-PA as cationic polyelectrolyte. On the other hand, the CG or polyvinyl sulfonic (PVS) acids were used as anionic polyelectrolytes. The films were prepared with PANI or PANI-PA intercalated with CG or with PVS alternately resulting in four films with different sequences: PANI/CG PANI-PA/CG, PANI/PVS and PANI-PA/PVS, respectively. Analysis by cyclic voltammetry (CV) of the films showed that the presence of gum increases the stability of the films in acidic medium. The performance of the modified electrode of PANI-PA/CG was evaluated in electro analytical determination of dopamine (DA). The tests showed great sensitivity of the film for this analyte that was detected at 10(-5) mol L(-1).

Development of a novel biosensor using cationic antimicrobial Peptide and nickel phthalocyanine ultrathin films for electrochemical detection of dopamine.

The antimicrobial peptide dermaseptin 01 (DS 01), from the skin secretion of Phyllomedusa hypochondrialis frogs, was immobilized in nanostructured layered films in conjunction with nickel tetrasulfonated phthalocyanines (NiTsPc), widely used in electronic devices, using layer-by-layer technique. The films were used as a biosensor to detect the presence of dopamine (DA), a neurotransmitter associated with diseases such as Alzheimer's and Parkinson's, with detection limits in the order of 10(-6) mol L(-1). The use of DS 01 in LbL film generated selectivity in the detection of DA despite the presence of ascorbic acid found in biological fluids. This work is the first to report that the antimicrobial peptide and NiTsPc LbL film exhibits electroanalytical activity to DA oxidation. The selectivity in the detection of DA is a fundamental aspect for the development of electrochemical sensors with potential applications in the biomedical and pharmaceutical industries.

Study of antimicrobial activity and atomic force microscopy imaging of the action mechanism of cashew tree gum.

The aim of this work was to evaluate the antimicrobial potential of two grades of cashew tree gum (crude and purified) against eight microorganisms and to analyze the mechanism of cashew tree gum antimicrobial action via atomic force microscopy (AFM) imaging. The results indicated strong antimicrobial properties of pure cashew tree gum against all tested microorganisms, except for Candida albicans and Lactobacillus acidophilus. On the other hand crude cashew gum showed antimicrobial activity only against Gram-positive bacteria (MRSA, MSSA, Listeria innocua and Enterococcus faecium). Atomic force microscopy imaging showed that pure cashew tree gum lead to bacterial cell collapse. In conclusion cashew tree gum presented relevant antimicrobial activity against most of the studied bacteria, and the purification of the cashew gum affected its antimicrobial spectrum.

Natural polysaccharides as active biomaterials in nanostructured films for sensing.

The search for natural, biocompatible and degradable materials amenable to be used in biomedical/analytical applications has attracted attention, either from the environmental or medical point of view. Examples are the polysaccharides extracted from natural gums, which have found applications in the food and pharmaceutical industries as stabilizers or thickening agent. In a previous paper, however, it was shown that a Brazilian natural gum, chicha (Sterculia striata), is suitable for application as building block for nanostructured film fabrication in conjunction with phthalocyanines. The films displayed electroactivity and could be used in sensing. In the present paper, we introduce the use of two different natural gums, viz., angico (Anadenanthera colubrina) and caraia (Sterculia urens), as active biomaterials to be used to modification layers, in the form of nanostructured thin films, including the study of dopamine detection. The multilayer films were assembled in conjunction with nickel tetrasulfonated phthalocyanines (NiTsPC) and displayed good chemical and electrochemical stability, allowing their use as transducer elements in sensors for detection of specific neurotransmitters. It is suggested here that nanoscale manipulation of new biodegradable natural polymers opens up a variety of new opportunities for the use of these materials in advanced biomedical and analytical devices.

Leishmanicidal activity and immobilization of dermaseptin 01 antimicrobial peptides in ultrathin films for nanomedicine applications.

Antimicrobial peptides (AMPs) are essential for the innate immune system of eukaryotes, imparting protection against pathogens and their proliferation in host organisms. The recent interest in AMPs as active materials in bionanostructures is due to the properties shown by these biological molecules, such as the presence of an alpha-helix structure and distribution of positive charges along the chain. In this study the antimicrobial peptide dermaseptin 01 (DS 01), from the skin secretion of Phyllomedusa hypochondrialis frogs was immobilized in nanostructured layered films in conjunction with nickel tetrasulfonated phthalocyanines. The leishmanicidal activity of DS 01 was confirmed using kinetic essays, in which DS 01 promoted death of all metacyclic promastigote cells in 45 minutes. Surprisingly, the immobilized DS 01 molecules displayed electroactivity, as revealed by electrochemical experiments, in which an oxidation peak at about 0.61 V was observed for a DS 01 monolayer deposited on top of a conductive electrode. Such electroactivity was used to investigate the sensing abilities of the nanostructured films toward Leishmania. We observed an increase in the oxidation current as a function of number of Leishmania cells in the electrolytic solution at concentrations down to 10(3) cells/mL. The latter is indicative that the use of AMPs immobilized in electroactive nanostructured films may be of interest for applications in the pharmaceutical industry and diagnosis. FROM THE CLINICAL EDITOR: The recent interest in Antimicrobial peptides (AMPs) as active materials in bionanostructures is due to the properties shown by these biological molecules. Leishmanicidal activity of a particular AMP is demonstrated in this paper.

Natural gum-assisted phthalocyanine immobilization in electroactive nanocomposites: physicochemical characterization and sensing applications.

Natural gums have been traditionally applied in cosmetics and the food industry, mainly as emulsification agents. Due to their biodegradability and excellent mechanical properties, new technological applications have been proposed involving their use with conventional polymers forming blends and composites. In this study, we take advantage of the polyelectrolyte character exhibited by the natural gum ChichA (Sterculia striata), extracted in the Northeastern region of Brazil, to produce electroactive nanocomposites. The nanocomposites were fabricated in the form of ultrathin films by combining a metallic phthalocyanine (nickel tetrasulfonated phthalocyanine, NiTsPc) and the ChichA gum in a tetralayer architecture, in conjunction with conventional polyelectrolytes. The presence of the gum led to an efficient adsorption of the phthalocyanine and enhanced the electrochemical response of the films. Upon combining the electrochemical and UV-vis absorption data, energy diagrams of the ChichA/NiTsPc-based system were obtained. Furthermore, modified electrodes based on gum/phthalocyanine films were able to detect dopamine at concentrations as low as 10-5 M.