Advantages of stereolithographic 3D printing in the fabrication of the Affiblot device for dot-blot assays.

In stereolithographic (SLA) 3D printing, objects are constructed by exposing layers of photocurable resin to UV light. It is a highly user-friendly fabrication method that opens a possibility for technology sharing through CAD file online libraries. Here, we present a prototyping procedure of a microfluidics-enhanced dot-blot device (Affiblot) designed for simple and inexpensive screening of affinity molecule characteristics (antibodies, oligonucleotides, cell receptors, etc.). The incorporation of microfluidic features makes sample processing user-friendly, less time-consuming, and less laborious, all performed completely on-device, distinguishing it from other dot-blot devices. Initially, the Affiblot device was fabricated using CNC machining, which required significant investment in manual post-processing and resulted in low reproducibility. Utilization of SLA 3D printing reduced the amount of manual post-processing, which significantly streamlined the prototyping process. Moreover, it enabled the fabrication of previously impossible features, including internal fluidic channels. While 3D printing of sub-millimeter microchannels usually requires custom-built printers, we were able to fabricate microfluidic features on a readily available commercial printer. Open microchannels in the size range 200-300 µm could be fabricated with reliable repeatability and sealed with a replaceable foil. Economic aspects of device fabrication are also discussed.

Acoustofluidic Properties of Polystyrene Microparticles.

Acoustophoresis has become a powerful tool to separate microparticles and cells, based on their material and biophysical properties, and is gaining popularity in clinical and biomedical research. One major application of acoustophoresis is to measure the compressibility of cells and small organisms, which is related to their contents. The cell compressibility can be extracted from the acoustic mobility, which is the main output of acoustic migration experiments, if the material properties and sizes of reference particles, the size of the cells, and the surrounding medium are known. Accurate methods to measure and calibrate the acoustic energy density in acoustophoresis systems are therefore critical. In this Perspective, polystyrene microparticles have become the most commonly used reference particles in acoustophoresis, due to their similar biophysical properties to cells. We utilized a two-step focusing method to measure the relative acoustic mobility of polystyrene beads of various sizes and colors and present a quantitative analysis of the variation in acousto-mechanical properties of polystyrene microparticles, showing a large spread in their material properties. A variation of more than 25% between different particle types was found. Thus, care is required when relying on polystyrene particles as a reference when characterizing acoustofluidics systems or acousto-mechanical properties of cells.

Acoustofluidic platforms for particle manipulation.

There is an increasing interest in acoustics for microfluidic applications. This field, commonly known as acoustofluidics involves the interaction of ultrasonic standing waves with fluids and dispersed microparticles. The combination of microfluidics and the so-called acoustic standing waves (ASWs) led to the development of integrated systems for contact-less on-chip cell and particle manipulation where it is possible to move and spatially localize these particles based on the different acoustophysical properties. While it was initially suggested that the acoustic forces could be harmful to the cells and could impact cell viability, proliferation, or function via phenotypic or even genotypic changes, further studies disproved such claims. This review is summarizing some interesting applications of acoustofluidics in the manipulations of biomaterials, such as cells or subcellular vesicles, in works published mainly within the last 5 years.

Macrofluidic Device for Preparative Concentration Based on Epitachophoresis.

We have developed a new separation device to concentrate and collect ions from several milliliter sample volumes to microliter fractions. Unlike most conventional platforms, this device has circular architecture. The electrophoretic migration operates from the outer perimeter toward the center. Separations can be performed both in continuous (zone electrophoresis) and discontinuous (moving boundary) electrolyte systems. We use a discontinuous electrolyte system comprising a leading and a terminating electrolyte to concentrate samples containing small organic anions and DNA fragment. The agarose gel stabilizes the boundary between the leading and terminating electrolytes. The milliliter volume sample is mixed with the terminating electrolyte and migrates through the gel toward the center. The concentrated total sample is collected in microliter fraction at the center. The potential for preparative concentration of DNA is demonstrated using a DNA ladder. Because zone migration accelerates as it moves toward the center, we named this method Epitachophoresis from the Greek word "επιταχυνω (epitachýnο)", meaning "acceleration". To the best of our knowledge, this unique circular architecture has not been previously described.

Fluid manipulation on the micro-scale: Basics of fluid behavior in microfluidics.

Fluid manipulation on the micro-scale (microfluidics) is bringing new potential applications in a number of fields, including chemistry, biology and medicine. At sub-millimeter channel scale, some phenomena, unimportant at the macroscale, become an important force to consider when designing a microfluidics system. For example, the decrease in fluid mass causes the effects of viscosity to overcome the influence of inertia. Turbulent flow cannot be achieved at any realistic fluid velocity, making mixing a challenging task. The only phenomenon capable of blending liquids at microscale is diffusion and liquid streams can be flowed side-by-side for tens of minutes before they completely fuse together. The decrease in the channel size also leads to an increased surface-to-volume ratio, which increases the importance of surface effects, including adsorption, capillary action and surface wetting and/or electric double layer formation with related electrokinetic phenomena. While rivers cannot flow uphill, a stream of liquid can easily flow up against gravity inside a capillary. Similarly, the formation of electric double layer near the charged surface of a micro-channel or capillary can be applied for electrokinetic actuating. This review summarizes selected physical phenomena related to liquid-based (water solutions) microfluidics as described recently.

Relative asymptotic oscillations of the out-of-time-ordered correlator as a quantum chaos indicator.

A detailed numerical study reveals that the asymptotic values of the standard-deviation-to-mean ratio of the out-of-time-ordered correlator in energy eigenstates can be successfully used as a measure of the quantum chaoticity of the system. We employ a finite-size fully connected quantum system with two degrees of freedom, namely, the algebraic u(3) model, and demonstrate a clear correspondence between the energy-smoothed relative oscillations of the correlators and the ratio of the chaotic part of the volume of phase space in the classical limit of the system. We also show how the relative oscillations scale with the system size and conjecture that the scaling exponent can also serve as a chaos indicator.

Effect of Different Feed Particle Size on Gastrointestinal Tract Morphology, Ileal Digesta Viscosity, and Blood Biochemical Parameters as Markers of Health Status in Broiler Chickens.

The study is focused on how the physical structure of the feed affects the health status of broiler chickens. The aim of this study was to evaluate the influence of feed particle size in broiler diets on gastrointestinal tract morphology, digesta viscosity, and blood biochemical parameters. A total of 90 one-day-old male Ross 308 broiler chickens were randomly divided into three different experimental groups (with five replicates per pen), with 6 birds per cage. The first experimental group (Coarse) was fed with the coarsest particle size, with feed with a geometric mean diameter (GMD) of 1111.26 µm, the next group (Medium) was fed with a less coarse feed size of GMD 959.89 µm, and the last group (Fine) was fed a diet with a fine feed particle size of GMD 730.48 µm. The use of coarse feed particle size in the diet had a positive effect on the gizzard weight and small intestinal villi height and crypt depth, which increased the surface area intended for digesting nutrients. The use of finely ground particles in the feed increased the level of gamma-glutamyl transferase and at the same time, decreased the level of urea, which could indicate adverse changes in the liver.

Caraway (Carum carvi L.) in fast-growing and slow-growing broiler chickens' diets and its effect on performance, digestive tract morphology and blood biochemical profile.

The aim of this study was to evaluate the addition of caraway (1%) in fast-growing and slow-growing broiler chickens' diet and its effect on performance parameters, blood biochemical profile, and relative organ sizes and ileum morphology in slow-growing broilers. Two separated experiments were performed. On the first day of age, the broilers were divided into 2 equal groups (Control and Caraway) with 6 replicates per treatment in both experiments. Experiment I: The total of 276 male fast-growing Ross 308 broiler chickens were used. The trial lasted from the first day to 35th day of chickens' age. Experiment II: The total of 216 male slow-growing (Hubbard JA 57) broilers were used. The trial lasted from the first to 50th day of chickens' age. Mean liveweight, weight gain, feed conversion ratio, blood biochemical parameters, and relative organ sizes were not significantly different in these trials. The group of slow-growing broilers supplemented with 1% of caraway in the diet showed longer villi and deeper crypt in the ileum after 50 d of life. Based on our results, it can be stated that the proportion of 1% caraway in fast-growing and slow-growing broiler chickens' diet did not influence performance parameters, blood biochemical profile and relative organ sizes. In case of the experiment with the slow-growing broilers supplemented with caraway, a significant difference in the height of the villi and the depth of the crypts was found. Caraway can be included in the broiler chickens' diets without negative effects, but further study of the effect on the intestinal morphology is necessary.

3D printed device for epitachophoresis.

Polyacrylamide or agarose gels are the most frequently used sieving and stabilizing media in slab gel electrophoresis. Recently, we have introduced a new electrophoretic technique for concentration/separation of milliliter sample volumes. In this technique, the gel is used primarily as an anticonvection media eliminating liquid flow during the electromigration. While serving well for the liquid stabilization, the gels can undergo deformation when exposed to a discontinuous electrolyte buffer system used in epitachophoresis. In this work, we have explored 3D printing to form rigid stabilizing manifolds to minimize liquid flow during the epitachophoresis run. The whole device was printed using the stereolithography technique from a low water-absorbing resin. The stabilizing manifold, serving as the gel substitute, was printed as a replaceable composite structure preventing electrolyte mixing during the separation. Different geometries of the 3D printed stabilizing manifolds were tested for use in concentrating ionic sample components without spatial separation. The presented device can focus analytes from 3 or 4 mL of the sample to 150 µL or less, depending on the collection cup size. With the 150 µL collection cup, this represents the enrichment factor from 20 to 27. The time of concentration was from 15 to 25 min, depending on stabilization media and power used.

Affiblot: a dot blot-based screening device for selection of reliable antibodies.

The key factor in the development of antibody-based assays is to find an antibody that has an appropriate affinity, high specificity, and low cross-reactivity. However, this task is not easy to carry out since the research antibodies on the market may suffer from low specificity and reproducibility. Here, we report on a palm-sized dot blot-based device, called the affiblot, that has a specially designed lid that allows simultaneous semi-quantitative comparison of up to five antibodies from different suppliers regarding their affinity/avidity, cross-reactivity, and batch-to-batch reliability. The only required peripheral equipment is a vacuum pump, a camera, and densitometry software. The affiblot device was tested for its functionality and its measurements were compared against those obtained by standard dot blot and ELISA. The benefit over these methods, when various antibodies are evaluated, is in its simplicity. It allows easy antigen deposition, fast application and the discarding of the solutions, a compact undivided membrane, and therefore significant decrease of labor. The device was tested with specific anti-ApoE, anti-EpCAM, anti-Salmonella, anti-E. coli, and anti-Listeria antibodies from different suppliers. Their properties were compared for their ability to interact specifically with antigen and/or non-target structures and the best-suited antibody for the intended application was identified.

Safety of Mealworm Meal in Layer Diets and their Influence on Gut Morphology.

The main objective of this study was to verify the safety of mealworm meal in the feed of laying hens from 17 to 42 weeks of age. Therefore, the feed mixtures were tested in terms of microbiological stability, fungal and mycotoxin content and selected parameters of hens' intestinal morphology and physiology were monitored. The experiment was carried out with 30 Lohmann Brown Classic hens. Hens were divided by body mass into three equal groups with 10 replicates per treatment. The two experimental groups received feed mixtures containing 2% and 5% yellow mealworm (Tenebrio molitor L.) meal. The third group was a control group which had 0% of mealworm meal in the diet. Diets with 2% and 5% of mealworm meals did not affect the length of villi and microbiome of the caecum. The highest digesta viscosity from the ileum was found in the group with 5% mealworm, which may indicate a slower passage of the digesta through the digestive tract. Based on our results, it may be concluded that the proportion of mealworm meals does not deteriorate the quality of feeds. Mealworm meal does not negatively affect microbial stability in experimental feeds. Therefore, it can be recommended the two and (or) five percent of mealworm meal inclusion in hen's diet.

Transkarbams as transdermal permeation enhancers: effects of ester position and ammonium carbamate formation.

Transkarbam 12, an ammonium carbamate formed by the reaction of dodecyl 6-aminohexanoate with carbon dioxide, is a highly active, broad-spectrum, nontoxic, and nonirritant transdermal permeation enhancer. It probably acts by a dual mechanism: a part of its activity is associated with the carbamic acid salt and/or its decomposition in the acidic stratum corneum. The ammonium ester thereby released is an active enhancer species as well, and its activity highly depends on the position of the ester group.

Dicarboxylic acid esters as transdermal permeation enhancers: effects of chain number and geometric isomers.

A series of transdermal permeation enhancers based on dicarboxylic acid esters was studied. Single-chain amphiphiles were markedly more effective than the double-chain ones. Monododecyl maleate, that is a cis derivative, was a more potent enhancer than its trans isomer, while the activity of succinates strongly depended on the donor vehicle. No difference between diastereoisomeric tartaric and meso-tartaric acid derivatives was found.

Synthesis of fluorescent C24-ceramide: evidence for acyl chain length dependent differences in penetration of exogenous NBD-ceramides into human skin.

Topical skin lipid supplementation may provide opportunities for controlling ceramide (Cer) deficiency in skin diseases such as atopic dermatitis or psoriasis. Here we describe the synthesis of a long-chain 7-nitrobenzo[c][1,2,5]oxadiazol-4-yl (NBD)-labeled Cer and its different penetration through human skin compared to widely used short-chain fluorescent Cer tools.

Simple Fabrication of Structured Magnetic Metallic Nano-Platelets for Bio-Analytical Applications.

This short communication presents a simple method of preparation of thin-metal nano-platelets utilizing metal sputtering and lift-off photolithography. The method offers complete control over size, shape and properties of nano-platelets of sub-micrometer thickness. Platelets with a thickness of 50⁻200 nm and with defined arbitrary shapes and sizes in the range of 15⁻300 µm were prepared from single or multiple metal layers by magnetron sputtering. Deposition of different metals in layers enabled fabrication of bi- or tri-metallic platelets with a magnetic core and differently composed surfaces. Highly reflective nano-platelets with a magnetic core allowed manipulation by magnetic fields, while different metallic surfaces served for functionalization by selected molecules. Submicron thin nano-platelets are extremely light (e.g., ~20 ng for a 100 µm × 100 µm × 0.1 µm gold nano-platelet) so that they can be attached to surfaces by only a few chemical bonds. At the same time their area is sufficiently large for simple optical recognition of their shape which is intended to label various characteristics depending on the specific surface functionalization of the given shape.

Transkarbams with terminal branching as transdermal permeation enhancers.

Transkarbams (T) represent novel group of highly active, non-toxic transdermal permeation enhancers. This study was focused on the influence of small symmetrical terminal branching on their enhancing activity. Series of T with terminal methyl or ethyl branching was prepared and their permeation-enhancing activity was compared to that of their linear analogues. The results showed completely a different behaviour from similarly branched alcohols, supporting the key role of the ammonium-carbamate polar head in the enhancing activity of T.

Transdermal and dermal delivery of adefovir: effects of pH and permeation enhancers.

The objective of this work was to investigate feasibility of transdermal and dermal delivery of adefovir (9-(2-phosphonomethoxyethyl)adenine), a broad-spectrum antiviral from the class of acyclic nucleoside phosphonates. Transport of 2% adefovir through and into porcine skin and effects of various solvents, pH, and permeation enhancers were studied in vitro using Franz diffusion cell. From aqueous donor samples, adefovir flux through the skin was 0.2-5.4 microg/cm2/h with greatest permeation rate at pH 7.8. The corresponding adefovir skin concentrations reached values of 120-350 microg/g of tissue. Increased solvent lipophilicity resulted in higher skin concentration but had only minor effect on adefovir flux. A significant influence of counter ions on both transdermal and dermal transport of adefovir zwitterion was observed at pH 3.4. Permeation enhancer dodecanol was ineffective, 1-dodecylazepan-2-one (Azone) and dodecyl 2-(dimethylamino)propionate (DDAIP) showed moderate activity. The highest adefovir flux (11.3+/-3.6 microg/cm2/h) and skin concentration (1549+/-416 microg/g) were achieved with 1% Transkarbam 12 (5-(dodecyloxycarbonyl)pentylammonium 5-(dodecyloxycarbonyl)pentylcarbamate) at pH 4. This study suggests that, despite its hydrophilic and ionizable nature, adefovir can be successfully delivered through the skin.

Permeation enhancer dodecyl 6-(dimethylamino)hexanoate increases transdermal and topical delivery of adefovir: influence of pH, ion-pairing and skin species.

Adefovir (9-(2-phosphonomethoxyethyl)adenine) is an acyclic nucleoside phosphonate currently used for the treatment of hepatitis B. The aim of this study was to evaluate the effect of permeation enhancer DDAK (6-dimethylaminohexanoic acid dodecyl ester) on the transdermal and topical delivery of adefovir. In porcine skin, DDAK enhanced adefovir flux 42 times with maximum at pH 5.8 suggesting ion pair formation. DDAK increased thermodynamic activity and stratum corneum/vehicle distribution coefficient of adefovir, as well as it directly decreased the skin barrier resistance. Maximal flux was observed already at 2% adefovir+1% DDAK. The results were confirmed in freshly excised human skin where DDAK enhanced adefovir flux 179 times to 8.9 microg/cm(2)/h. This rate of percutaneous absorption would allow for reaching effective plasma concentrations. After the topical application, adefovir concentrated in the stratum corneum with low penetration into the deeper skin layers from either aqueous or isopropyl myristate vehicle without the enhancer. With 1% DDAK, adefovir concentrations in the viable epidermis and dermis were 33-61 times higher. These results offer an attractive alternative to established routes of administration of adefovir and other acyclic nucleoside phosphonates.

HPLC method for determination of in vitro delivery through and into porcine skin of adefovir (PMEA).

A simple HPLC/UV method for the determination of the transdermal permeation and dermal penetration of a broad-spectrum antiviral drug adefovir (PMEA) was developed. The separation was achieved on a C18 column with the mobile phase composed of 10 mM KH2PO4 and 2 mM Bu4NHSO4 at pH 6.0 and 7% acetonitrile. The method was validated with respect to selectivity, linearity (0.1-50 microg/ml), precision, accuracy, and stability. Transdermal permeation of 2% PMEA was studied in vitro using the Franz diffusion cell and porcine skin. The flux values were 1.8, 3.0, and 0.6 microg/cm2/h from aqueous donor samples at pH 3.4 and 7.4, and isopropyl myristate, respectively. The respective skin concentrations at 48 h were 294, 263, and 971 microg/g from these vehicles. These results will serve as a lead for further studies on transdermal and topical delivery of antivirals from the group of acyclic nucleoside phosphonates.

Rapid and simple preparation of thiol-ene emulsion-templated monoliths and their application as enzymatic microreactors.

A novel, rapid and simple method for the preparation of emulsion-templated monoliths in microfluidic channels based on thiol-ene chemistry is presented. The method allows monolith synthesis and anchoring inside thiol-ene microchannels in a single photoinitiated step. Characterization by scanning electron microscopy showed that the methanol-based emulsion templating process resulted in a network of highly interconnected and regular thiol-ene beads anchored solidly inside thiol-ene microchannels. Surface area measurements indicate that the monoliths are macroporous, with no or little micro- or mesopores. As a demonstration, galactose oxidase and peptide-N-glycosidase F (PNGase F) were immobilized at the surface of the synthesized thiol-ene monoliths via two different mechanisms. First, cysteine groups on the protein surface were used for reversible covalent linkage to free thiol functional groups on the monoliths. Second, covalent linkage was achieved via free primary amino groups on the protein surface by means of thiol-ene click chemistry and l-ascorbic acid linkage. Thus prepared galactose oxidase and PNGase F microreactors demonstrated good enzymatic activity in a galactose assay and the deglycosilation of ribonuclease B, respectively.