Peculiarities of Integrating Mechanical Valves in Microfluidic Channels Using Direct Laser Writing.

Regenerative medicine is a fast expanding scientific topic. One of the main areas of development directions in this field is the usage of additive manufacturing to fabricate functional components that would be later integrated directly into the human body. One such structure could be a microfluidic valve which could replace its biological counterpart in veins as it is worn out over the lifetime of a patient. In this work, we explore the possibility to produce such a structure by using multiphoton polymerization (MPP). This technology allows the creation of 3D structures on a micro- and nanometric scale. In this work, the fabrication of microfluidic systems by direct laser writing was carried out. These devices consist of a 100 µm diameter channel and within it a 200 µm long three-dimensional one-way mechanical valve. The idea of this device is to have a single flow direction for a fluid. For testing purposes, the valve was integrated into a femtosecond laser-made glass microfluidic system. Such a system acts as a platform for testing such small and delicate devices. Measurements of the dimensions of the device within such a testing platform were taken and the repeatability of this process was analyzed. The capability to use it for flow direction control is measured. Possible implications to the field of regenerative medicine are discussed.

Fabrication of Microfluidic Tesla Valve Employing Femtosecond Bursts.

Expansion of the microfluidics field dictates the necessity to constantly improve technologies used to produce such systems. One of the approaches which are used more and more is femtosecond (fs) direct laser writing (DLW). The subtractive model of DLW allows for directly producing microfluidic channels via ablation in an extremely simple and cost-effective manner. However, channel surface roughens are always a concern when direct fs ablation is used, as it normally yields an RMS value in the range of a few µm. One solution to improve it is the usage of fs bursts. Thus, in this work, we show how fs burst mode ablation can be optimized to achieve sub-µm surface roughness in glass channel fabrication. It is done without compromising on manufacturing throughput. Furthermore, we show that a simple and cost-effective channel sealing methodology of thermal bonding can be employed. Together, it allows for production functional Tesla valves, which are tested. Demonstrated capabilities are discussed.

Analysis of the Leaves and Cones of Lithuanian Hops (Humulus lupulus L.) Varieties by Chromatographic and Spectrophotometric Methods.

This work involves a comprehensive chemical composition analysis of leaf and cone samples of Lithuanian hop varieties. This study aimed to determine the chemometric properties of the leaves and cones of five Lithuanian hop varieties. Determined properties were the following: (a) xanthohumol content, (b) phenolic compounds, (c) flavonoids, (d) radical scavenging activity, and (e) the qualitative composition of volatile compounds. The total content of phenolic compounds in aqueous 75% methanolic extracts varied between 31.4-78.2 mg of rutin equivalents (RE)/g, and the concentration of flavonoids was between 11.0-23.3 mg RE/g. Radical scavenging activity varied between 34.4-87.2 mg RE/g. A QUENCHER analysis procedure showed 91.7-168.5 mg RE/g of the total phenolic compound content, 12.7-21.4 mg RE/g of flavonoids, and 48.4-121.0 mg RE/g of radical scavenging activity. 'Fredos taurieji' and 'Fredos derlingieji' varieties have shown maximum values of phenolic compounds and radical scavenging activity both in leaf and cone suspensions. These varieties accumulated a higher amount of xanthohumol in leaves. The concentration of xanthohumol in the samples varied between 0.0014-0.2136% of dry mass, with the highest concentration in the cones of 'Kauno grazieji'. We identified 19 volatile compounds in leaves, and in cones, we identified 32. In both of them, α-humulene and ß caryophyllene dominated. 'Raudoniai' leaves were exceptional in their aroma due to dominating compound nagina ketone (Kovats index 1306). The QUENCHER procedure has shown a great potential for the unextractable residue of hop raw material. Further investigation and valorization of different hop biomass components, not only cones, are essential.

Mathematical Model Coupled to Neural Networks Calculates the Extraction Recovery of Polycyclic Aromatic Hydrocarbons in Problematic Matrix.

Unknown extraction recovery from solid matrix samples leads to meaningless chemical analysis results. It cannot always be determined, and it depends on the complexity of the matrix and properties of the extracted substances. This paper combines a mathematical model with the machine learning method-neural networks that predict liquid extraction recovery from solid matrices. The prediction of the three-stage extraction recovery of polycyclic aromatic hydrocarbons from a wooden railway sleeper matrix is demonstrated. Calculation of the extraction recovery requires the extract's volume to be measured and the polycyclic aromatic hydrocarbons' concentration to be determined for each stage. These data are used to calculate the input values for a neural network model. Lowest mean-squared error (0.014) and smallest retraining relative standard deviation (20.7%) were achieved with the neural network setup 6:5:5:4:1 (six inputs, three hidden layers with five, five, and four neurons in a layer, and one output). To train such a neural network, it took less than 8000 steps-less than a second--using an average-performance laptop. The relative standard deviation of the extraction recovery predictions ranged between 1.13 and 5.15%. The three-stage recovery of the extracted dry sample showed 104% of three different polycyclic aromatic hydrocarbons. The extracted wet sample recovery was 71, 98, and 55% for phenanthrene, anthracene, and pyrene, respectively. This method is applicable in the environmental, food processing, pharmaceutical, biochemical, biotechnology, and space research areas where extraction should be performed autonomously without human interference.

Evaluation of Chemical Composition, Radical Scavenging and Antitumor Activities of Satureja hortensis L. Herb Extracts.

Satureja hortensis L. is an annual herbaceous plant of the Lamiaceae Lindl. family. S. hortensis L., related to thyme and rosemary, is used as spice and traditional medicinal herb in Europe. Mainly due to the polyphenols contained in S. hortensis L., this plant exhibits multiple biological effects. However, therapeutic effects on cells, including skin tumors, have not yet been studied. Therefore, the aim of this study was to compare the composition and the resulting antioxidant as well as biological properties [on melanocytes and melanoma cells] of summer, savory growing in botanical garden of Vytautas Magnus University in middle Lithuania climatic conditions, collected during various phases of vegetation. It has been shown that the budding phase alcohol extract of this plant contains the largest amounts of polyphenols, including rutin and rosemary acid, which promote the radical scavenging activity and antioxidant properties. In contrast, the extract from the massive flowering phase already at a concentration of 12.5 µg/mL reduces the survival of melanoma cells to 60% with 90% melanocytes survival. In addition, extracts from beginning of flowering and end of flowering at a concentration of 25 µg/mL, containing significantly less rutin and rosmarinic acid, in combination with irradiation of cells with UVB, significantly increased the lipid peroxidation process, particularly in melanoma cells. These data indicate the possibility of using extracts from S. hortensis L. to modulate/differentiate the metabolism of normal and tumor skin cells.

Single-chip based contactless conductivity detection system for multi-channel separations.

In this work, the design and characterization of a multi-cell capacitively coupled contactless conductivity detection system are described. The operation and simultaneous acquisition from 3 detector cells are demonstrated, however, the system is capable of supplying 8 detection cells and can be easily upgraded to maintain 64 capacitively coupled contactless conductivity detection cells. On performing flow-injection analysis, the system recorded as low as 0.01 mM of acetic acid, phosphoric acid, NaH2PO4, and Na2B4O7 solutions in water. The instrument was also capable of recording and distinguishing different mixtures of organic solvents: (a) methanol-acetonitrile, (b) hexane-acetone. The designed detection system is expected to be used coupled with multi-channel separation devices for monitoring simultaneous processes.

Chromatographic Data Segmentation Method: A Hybrid Analytical Approach for the Investigation of Antiviral Substances in Medicinal Plant Extracts.

The methodology described in this article will significantly reduce the time required for understanding the relations between chromatographic data and bioactivity assays. The methodology is a hybrid of hypothesis-based and data-driven scientific approaches. In this work, a novel chromatographic data segmentation method is proposed, which demonstrates the capability of finding what volatile substances are responsible for antiviral and cytotoxic effects in the medicinal plant extracts. Up until now, the full potential of the separation methods has not been exploited in the life sciences. This was due to the lack of data ordering methods capable of adequately preparing the chromatographic information. Furthermore, the data analysis methods suffer from multidimensionality, requiring a large number of investigated data points. A new method is described for processing any chromatographic information into a vector. The obtained vectors of highly complex and different origin samples can be compared mathematically. The proposed method, efficient with relatively small sized data sets, does not suffer from multidimensionality. In this novel analytical approach, the samples did not need fractionation and purification, which is typically used in hypothesis-based scientific research. All investigations were performed using crude extracts possessing hundreds of phyto-substances. The antiviral properties of medicinal plant extracts were investigated using gas chromatography-mass spectrometry, antiviral tests, and proposed data analysis methods. The findings suggested that (i) ß- cis-caryophyllene, linalool, and eucalyptol possess antiviral activity, while (ii) thujones do not, and (iii) α-thujone, ß-thujone, cis- p-menthan-3-one, and estragole show cytotoxic effects.

Evaluation of Fresh Cheese Quality Prepared with Newly Isolated Nisin Z-Producing Lactococcus lactis Bacteria.

The main task of the present study was to evaluate an impact of three nisin Z-producing Lactococcus lactis bacteria newly isolated from raw goat milk for some fresh cow cheese characteristics during the storage. Microbiological evaluation for Listeria monocytogenes, Staphylococcus aureus, and viable lactic acid bacteria counts and determination of pH, titratable acidity, and lactic acid concentration of produced cheese were performed after 0, 24, 48, 72, and 96 h. Sensory analysis for the evaluation of acidity, flavor intensity, color intensity, bitterness, and crumbliness of prepared cheese was performed. The changes of volatile compounds in fresh cheese were evaluated using headspace solid phase microextraction (SPME) coupled with gas chromatography-mass spectrometry. Chemometric methods were applied for the data analysis. Study showed that tested bacteria are suitable for the manufacturing of fresh cheese and possible application for fresh cheese biopreservation, as pathogenic bacteria did not grow during 4 days (96 h); chemometric analysis revealed that L. lactis strain LL56 was the most similar to commercially available L. lactis ATCC11454.

Optimization of a capillary zone electrophoresis-contactless conductivity detection method for the determination of nisin.

Determination of natural preservatives using electrophoretic or chromatographic techniques in fermented milk products is a complex task due to the following reasons: (i) the concentrations of the analytes can be below the detection limits, (ii) complex matrix and comigrating/coeluting compounds in the sample can interfere with the analytes of the interest, (iii) low recovery of the analytes, and (iv) the necessity of complex sample preparation. The aim of this study was to apply capillary zone electrophoresis coupled with contactless conductivity detection for the separation and determination of nisin in fermented milk products. In this work, separation and determination of natural preservative-nisin in fermented milk products is described. Optimized conditions using capillary zone electrophoresis coupled with capacitance-to-digital technology based contactless conductivity detector and data conditioning, which filter the noise of the electropherogram adaptively to the peak migration time, allowed precise, accurate, sensitive (limit of quantification: 0.02 µg/mL), and most importantly requiring very minute sample preparation, determination of nisin. Sample preparation includes following steps: (i) extraction/dilution and (ii) centrifugation. This method was applied for the determination of nisin in real samples, i.e. fermented milk products. The values of different nisin forms were ranging from 0.056 ± 0.003 µg/mL to 9.307 ± 0.437 µg/g.

Interaction of carboxylated CdSe/ZnS quantum dots with fish embryos: Towards understanding of nanoparticles toxicity.

Due to colloidal instability even with protective coatings, nanoparticles tend to aggregate in complex environments and possibly interact with biota. In this study, visualization of quantum dots (QDs) interaction with rainbow trout (Oncorhynchus mykiss) embryos was performed. Studies on zebrafish (Danio rerio) and pearl gourami (Trichogaster leerii) embryos have shown that QDs interact with embryos in a general manner and their affects are independent on the type of the embryo. It was demonstrated that carboxylated CdSe/ZnS QDs (4 nM) were aggregating in accumulation media and formed agglomerates on the surface of fish embryos under 1-12 days incubation in deep-well water. Detailed analysis of QDs distribution on fish embryos surface and investigation of the penetration of QDs through embryo's membrane showed that the chorion protects embryos from the penetration through the chorion and the accumulation of nanoparticles inside the embryos. Confocal microscopy and spectroscopy studies on rainbow trout embryos demonstrated that QDs cause chorion damage, due to QDs aggregation on the surface of chorion, even the formation of the agglomerates at the outer part of the embryos and/or with the mucus were detected. Aggregation of QDs and formation of agglomerates on the outer part of the embryo's membrane caused the intervention of the aggregates to the chorion and even partially destroyed the embryo's chorion. The incorporation of QDs in chorion was confirmed by two methods: in living embryos from a 3D reconstruction view, and in slices of embryos from a histology view. The damage of chorion integrity might have adverse effects on embryonic development. Moreover, for the first time the toxic effect of QDs was separated from the heavy metal toxicity, which is most commonly discussed in the literature to the toxicity of the QDs.

Current state of purification, isolation and analysis of bacteriocins produced by lactic acid bacteria.

The scientific interest for the search of natural means of microbial inhibitors has not faded for several years. A search of natural antibiotics, so-called bacteriocins which are produced by lactic acid bacteria (LAB), gains a huge attention of the scientists in the last century, in order to reduce the usage of synthetic food additives. Pure bacteriocins with wide spectra of antibacterial activity are promising among the natural biopreservatives. The usage of bacteriocin(s) producing LAB as starter culture for the fermentation of some food products, in order to increase their shelf-life, when synthetic preservatives are not allowable, is also possible. There are a lot of studies focusing on the isolation of new bacteriocins from traditional fermented food, dairy products and other foods or sometimes even from unusual non-food matrices. Bacteriocins producing bacteria have been isolated from different sources with the different antibacterial activity against food-borne microorganisms. This review covers the classification of bacteriocins, diversity of sources of bacteriocin(s) producing LAB, antibacterial spectra of isolated bacteriocins and analytical methods for the bacteriocin purification and analysis within the last 15 years.

Downscaling the in vitro test of fungal bioremediation of polycyclic aromatic hydrocarbons: methodological approach.

The miniaturization and optimization of a white rot fungal bioremediation experiment is described in this paper. The optimized procedure allows determination of the degradation kinetics of anthracene. The miniaturized procedure requires only 2.5 ml of culture medium. The experiment is more precise, robust, and better controlled comparing it to classical tests in flasks. Using this technique, different parts, i.e., the culture medium, the fungi, and the cotton seal, can be analyzed. A simple sample preparation speeds up the analytical process. Experiments performed show degradation of anthracene up to approximately 60% by Irpex lacteus and up to approximately 40% by Pleurotus ostreatus in 25 days. Bioremediation of anthracene by the consortium of I. lacteus and P. ostreatus shows the biodegradation of anthracene up to approximately 56% in 23 days. At the end of the experiment, the surface tension of culture medium decreased comparing it to the blank, indicating generation of surfactant compounds.

Screening of antioxidant activity and volatile compounds composition of Chamerion angustifolium (L.) Holub ecotypes grown in Lithuania.

Since biological activity of medicinal plants is dependent on cultivation area, climatic conditions, developmental stage, genetic modifications and other factors, it is important to study flora present in different growing sites and geographical zones. This study was focused on screening of antioxidant activity of C. angustifolium harvested in six different locations in Lithuania. The total contents of phenolic compounds, flavonoids and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity were evaluated by spectrophotometric methods. A correlation between radical scavenging activity and total phenolic compounds content was observed (correlation coefficient 0.98). HPLC with online post-column DPPH radical scavenging reaction detection was used for the separation of extracts. Oenothein B, rutin and one unidentified compound were predominant. Volatile compounds were analysed using solid-phase microextraction coupled with gas chromatography-mass spectrometry. Based on the analysis of volatiles, all samples were classified into two chemotypes: (I) with predominant α- and ß-caryophyllenes and (II) with predominant anethole.

Safety and quality parameters of ready-to-cook minced pork meat products supplemented with Helianthus tuberosus L. tubers fermented by BLIS producing lactic acid bacteria.

The aim of this study was to evaluate the influence of additives of Jerusalem artichoke (JA), fermented with P. acidilactici KTU05-7, P. pentosaceus KTU05-9, L. sakei KTU05-6, on the quality and safety parameters of ready - to cook - minced pork (RCMP). Fermented JA additives reduced pH of the meat products and decreased water holding capacity (WHC) from 2.01 till 2.93 %. Concentrations of biogenic amines in RCMP with additives of the lactic acid bacteria (LAB) - fermented JA were significantly lower comparing with control sample. The number of pathogenic bacteria in artificially contaminated meat samples was significantly reduced in case of LAB-fermented JA additives. The highest antimicrobial activity was obtained using P. acidilactici fermented JA additives. The amounts of microbial pathogens E. coli and Ent. faecalis, S. aureus and Streptococcus spp. were determined 3.41, 3.38, 3,96 and 4.74 log CFU/g correspondingly, whereas without LAB-fermented JA additives were 8.94, 7.75, 8.82 and 8.58 log CFU/g, correspondingly. A possibility to improve sensory properties (flavor) of RCMP using LAB fermented JA additives was investigated. The composition of volatile compounds of RCMP without additive and with LAB-fermented JA additives was analyzed using gas chromatography-mass spectrometry (GC-MS). The results of sensory evaluation of meat products supplemented with fermented JA additives revealed specific odor, which is pleasant and acceptable for consumers might be explainable that LAB-fermented JA additives have shown considerable differences mainly due to the accumulation of volatiles such as toluene, ethylbenzene, decane, undecane, 2 methyl undecane. N-morpholinomethyl-isopropyl-sulfide, 6-undecilamine and N,N-dimethyl-1-pentadecanamine were not determined in RCMP with LAB-fermented JA additives. The results obtained show, that P. acidilactici fermented JA 5 % additive is most suitable for the RCMP processing in order to prevent microbiological spoilage, increase volatile compounds and acceptability of the products.

Evaluation of phytochemical composition of fresh and dried raw material of introduced Chamerion angustifolium L. using chromatographic, spectrophotometric and chemometric techniques.

Due to the wide spectrum of biological activities, Chamerion angustifolium L. as medicinal plant is used for the production of food supplements. However, it should be kept in mind that quality (biological activity) of the herb depends on its geographic origin, the way of raw material preparation or extraction and chemotype. The purpose of this study was to evaluate and compare the compositions of volatile, non-volatile compounds and antioxidant activities of C. angustifolium grown in Kaunas Botanical Garden after the introduction from different locations in Lithuania. The compositions of fresh and air-dried samples were compared. The profile of volatile compounds was analyzed using headspace solid phase microextraction coupled with GC/MS. trans-2-Hexenal (16.0-55.9% of all volatiles) and trans-anethole (2.6-46.2%) were determined only in the dried samples, while cis-3-hexenol (17.5-68.6%) only in fresh samples. Caryophyllenes (α- and ß-) were found in all analyzed samples, contributing together from 2.4% to 52.3% of all volatiles according to the origin and preparation (fresh or dried) of a sample. Total amount of phenolic compounds, total content of flavonoids and radical scavenging activity (using 2,2-diphenyl-1-picrylhydrazyl (DPPH)) were determined using spectrophotometric assays. The variation of total phenolic compounds content was dependent on the sample origin, moreover, drying reduced amount of phenolics 1.5-3.5 times. The DPPH free radical scavenging activity was in the range of 238.6-557.1mg/g (expressed in rutin equivalents) in the fresh samples and drastically reduced to 119.9-124.8 mg/g after drying. The qualitative analysis of phenolic compounds in the aqueous methanolic extracts of C. angustifolium was performed by means of HPLC with UV detection. Oenothein B and rutin were predominant in the samples; also caffeic and chlorogenic acids, and quercetin were determined. Chemometric methods, namely principal component analysis, hierarchical cluster analysis and K-means clustering analysis, were applied for evaluation of the results. Chemometric analysis showed existence of different chemotypes of C. angustifolium L. and their relation to the geographic origin.

Comparative analysis of radical scavenging and antioxidant activity of phenolic compounds present in everyday use spice plants by means of spectrophotometric and chromatographic methods.

Comparative analysis of radical scavenging and antioxidant activities of phenolic compounds present in everyday use spice plants was carried out by means of spectrophotometric and chromatographic methods. Six spice plant samples, namely onion (Allium cepa), parsley (Petroselinum crispum) roots and leaves, celery (Apium graveolens) roots and leaves and leaves of dill (Anethum graveolens) were analyzed. Total amount of phenolic compounds and radical scavenging activity (RSA) was the highest in celery leaves and dill extracts and was the lowest in celery roots. Comparing commonly used spectrophotometric analysis of 2,2-diphenyl-1-picrylhydrazyl (DPPH) RSA of extracts with the results obtained using reversed-phase chromatographic separation with on-line post-column radical scavenging reaction detection, good correlation was obtained (R(2)=0.848). Studies using HPLC system with electrochemical detector showed that bioactive phytochemicals can be separated and antioxidant activities of individual compounds evaluated without the need of a complex HPLC system with reaction detector. The results obtained using electrochemical detection correlate with the RSA assayed using spectrophotometric method (R(2)=0.893).