Mechanochemical and Size Reduction Machines for Biorefining

In recent years, we have witnessed an increasing interest in the application of mechanochemical methods for processing materials in biomass refining techniques. Grinding and mechanical pretreatment are very popular methods utilized to enhance the reactivity of polymers and plant raw materials; however, the choice of devices and their modes of action is often performed through trial and error. An inadequate choice of equipment often results in inefficient grinding, low reactivity of the product, excess energy expenditure, and significant wear of the equipment. In the present review, modern equipment employing various types of mechanical impacts, which show the highest promise for mechanochemical pretreatment of plant raw materials, is examined and compared—disc mills, attritors and bead mills, ball mills, planetary mills, vibration and vibrocentrifugal mills, roller and centrifugal roller mills, extruders, hammer mills, knife mills, pin mills, disintegrators, and jet mills. The properly chosen type of mechanochemical activation (and equipment) allows an energetically and economically sound enhancement of the reactivity of solid-phase polymers by increasing the effective surface area accessible to reagents, reducing the amount of crystalline regions and the diffusion coefficient, disordering the supramolecular structure of the material, and mechanochemically reacting with the target substances.     

A miniaturized arrayed assay format for detecting small molecule-protein interactions in cells

Background: Two complementary approaches to studying the cellular function of proteins involve alteration of function either by mutating protein-encoding genes or by binding a small molecule to the protein. A mutagen can generate millions of genetic mutations; correspondingly, split-pool synthesis can generate millions of unique ligands attached to individual beads. Genetic screening of mutations is relatively straightforward but, in contrast, split-pool synthesis presents a challenge to current methods of screening for compounds that alter protein function. The methods used to screen natural products are not feasible for large libraries composed of covalently immobilized compounds on synthesis beads. The sheer number of compounds synthesized by split-pool synthesis, and the small quantity of individual compound attached to each bead require assay miniaturization for efficient screening.Results: We present a miniaturized cell-based technique for the screening of ligands prepared by split-pool synthesis. Spatially defined droplets with uniform volumes of approximately 50–150 nanoliters (depending on well dimensions) are arrayed on plastic devices prepared using a combination of photolithography and polymer molding. Using this microtechnology, approximately 6,500 assays using either yeast cells or mammalian tissue culture can be performed within the dimensions of a standard 10 cm petri dish. We demonstrate that the biological effect of a small molecule prepared by split-pool synthesis can be detected in this format following its photorelease from a bead.Conclusions: The miniaturized format described here allows uniformly sized nanodroplets to be arrayed on plastic devices. The design is amenable to a large number of biological assays and the spatially arrayed format ensures uniform and controlled ligand concentrations and should facilitate automation of assays. The screening method presented here provides an efficient means of rapidly screening large numbers of ligands made by split-pool synthesis in both yeast and mammalian cells.     

Ball milling in organic synthesis: solutions and challenges

During the last decade numerous protocols have been published using the method of ball milling for synthesis all over the field of organic chemistry. However, compared to other methods leaving their marks on the road to sustainable synthesis (e.g. microwave, ultrasound, ionic liquids) chemistry in ball mills is rather underrepresented in the knowledge of organic chemists. Especially, in the last three years the interest in this technique raised continuously, culminating in several high-quality synthetic procedures covering the whole range of organic synthesis. Thus, the present tutorial review will be focused on the highlights using this method of energy transfer and energy dissipation. The central aim is to motivate researchers to take notice of ball mills as chemical reactors, implementing this technique in everyday laboratory use and thus, pave the ground for future activities in this interdisciplinary field of research.     

On‐Bead Screens Sample Narrower Affinity Ranges of Protein–Ligand Interactions Compared to Equivalent Solution Assays

Conceptually, on‐bead screening is one of the most efficient high‐throughput screening (HTS) methods. One of its inherent advantages is that the solid support has a dual function: it serves as a synthesis platform and as a screening compartment. Compound purification, cleavage and storage and extensive liquid handling are not necessary in bead‐based HTS. Since the establishment of one‐bead one‐compound library synthesis, the properties of polymer beads in chemical reactions have been thoroughly investigated. However, the characterization of the kinetics and thermodynamics of protein–ligand interactions on the beads used for screening has received much less attention. Consequently, the majority of reported on‐bead screens are based on empirically derived procedures, independent of measured equilibrium constants and rate constants of protein binding to ligands on beads. More often than not, on‐bead screens reveal apparent high affinity binders through strong protein complexation on the matrix of the solid support. After decoding, resynthesis, and solution testing the primary hits turn out to be unexpectedly weak binders, or may even fall out of the detection limit of the solution assay. Only a quantitative comparison of on‐bead binding and solution binding events will allow systematically investigating affinity differences as function of protein and small molecule properties. This will open up routes for optimized bead materials, blocking conditions and other improved assay procedures. By making use of the unique features of our previously introduced confocal nanoscanning (CONA) method, we investigated the kinetic and thermodynamic properties of protein–ligand interactions on TentaGel beads, a popular solid support for on‐bead screening. The data obtained from these experiments allowed us to determine dissociation constants for the interaction of bead‐immobilized ligands with soluble proteins. Our results therefore provide, for the first time, a comparison of on‐bead versus solution binding thermodynamics. Our data indicate that affinity ranges found in on‐bead screening are indeed narrower compared to equivalent interactions in homogeneous solution. A thorough physico‐chemical understanding of the molecular recognition between proteins and surface bound ligands will further strengthen the role of on‐bead screening as an ultimately cost‐effective method in hit and lead finding.     

Saturated resins or stress of the resin

Although the solid-phase mode is an excellent strategy for the preparation of both biomolecules and small molecules, the synthesis of polyproline-based dendrimers has provided evidence that the capacity of the bead is limited. This phenomenon, which can be interpreted as saturation or stress of the resin, can lead to a complete breakdown of the bead structure.     

Patterned self-assembled beads in silicon channels.

A novel technique enabling selective bead trapping in microfluidic devices without the use of physical barriers is presented in this paper. It is a fast, convenient and simple method, involving microcontact printing and self-assembly, that can be applied to silicon, quartz or plastic substrates. In the first step, channels are etched in the substrate. The surface chemistry of the internal walls of the channels is then modified by microcontact printing. The chip is submerged in a bead slurry where beads self-assemble based on surface chemistry and immobilize on the internal walls of the channels. Silicon channels (100 microm wide and 50 microm deep) have been covered with monolayers of streptavidin-, amino- and hydroxy-functionalized microspheres and resulted in good surface coverage of beads on the channel walls. A high-resolution pattern of lines of self-assembled streptavidin beads, as narrow as 5 microm, has also been generated on the bottom of a 500 microm wide and 50 microm deep channel. Flow tests were performed in sealed channels with the different immobilized beads to confirm that the immobilized beads could withstand the forces generated by water flowing in the channels. The presented results indicate that single beads can be precisely positioned within microfluidic devices based on self-assembly which is useful as screening and analysis tools within the field of biochemistry and organic chemistry.     

Directed-sorting method for synthesis of bead-based combinatorial libraries of heterogeneous catalysts

The synthesis and analysis of inorganic material combinatorial libraries by a directed-sorting, split-pool bead method was demonstrated. Directed-sorting, split-pool, metal-loaded libraries were synthesized by adsorbing metal salts (H2PtCl6, SnCl2, CuCl2, and NiCl2) and metal standards (Pt, Cu, Ni in HCl) onto 2-mg porous gamma-alumina beads in 96- or 384-well plates. A matrix algorithm for the synthesis of bead libraries treated each bead as a member of a row or column of a given matrix. Computer simulations and manual tracking of the sorting process were used to assess library diversity. The bead compositions were analyzed by energy-dispersive X-ray spectroscopy, X-ray fluorescence spectroscopy, electron probe microanalysis, inductively coupled plasma atomic emission spectroscopy, and inductively coupled plasma mass spectroscopy. The metal-loaded beads were analyzed by laser-activated membrane introduction mass spectroscopy (LAMIMS) for catalytic activity using methylcyclohexane dehydrogenation to toluene as a probe reaction. The catalytic activity of individual beads that showed minimal (approximately 20% of that of Pt on alumina) to high conversion could be determined semiquantitatively by LAMIMS. This method, therefore, provides an alternative to screening using microreactors for reactors that employ catalysts in the form of beads. The directed-sorting method offers the potential for synthesis of focused libraries of inorganic materials through relatively simple benchtop split-pool chemistry.     

Nonthermal Plasma Synthesis of Nanocrystals: Fundamentals,Applications, and Future Research Needs

Nonthermal plasma synthesis has emerged as a viable alternative to nanocrystal synthesis in the liquid phase or by other gas phase based methods. The nonequilibrium environment containing free charge carriers enables the synthesis of nanocrystals with excellent crystallinity and narrow size distributions. This paper reviews the fundamental mechanisms involved in the synthesis of nanocrystals with nonthermal plasmas. It discusses the luminescent properties of plasma-produced silicon nanocrystals and their application in devices such as light emitting diodes. The ability of plasma synthesis to generate doped nanocrystals is a particularly appealing attribute. We present boron and phosphorous doped silicon nanocrystals and review their applications as near infrared plasmonic materials. Finally, the author presents his view of some important research needs in the area of nonthermal plasma synthesis of nanocrystals.     

A novel and rapid encoding method based on mass spectrometry for "one-bead-one-compound" small molecule combinatorial libraries

A novel and efficient encoding method based on mass spectrometry for "one-bead-one-compound" small molecule combinatorial libraries has been developed. The topologically segregated bifunctional resin beads with orthogonal protecting groups in the outer and inner regions are first prepared according to our previously published procedure. Prior to library synthesis, the inner core of each bead is derivatized with 3-4 different coding blocks on a cleavable linker. Each functional group on the scaffold is encoded by an individual coding block containing a functional group with the same chemical reactivity. During the library synthesis, the same chemical reactions take place on the scaffold (outer layer of the bead) and coding blocks (inner core of the bead) concurrently. After screening, the coding tags in the positive beads are released, followed by molecular mass determination using matrix-assisted laser desorption ionization Fourier transform mass spectrometry. The chemical structure of library compounds can be readily identified according to the molecular masses of the coding tags. The feasibility and efficiency of this approach were demonstrated by the synthesis and screening of a model small molecule library containing 84 672 member compounds, with a model receptor, streptavidin. Streptavidin binding ligands with structural similarity (17) were identified. The decoding results were clear and unambiguous.     

Software for automating analysis of encoded combinatorial libraries

This paper describes the applications which are used to automate the analysis of encoded combinatorial libraries. Commercial packages from MDL, Oracle and Agilent are linked with application software written in C/C++, in Microsoft Access and in ChemStation macro language. Encoding correspondence lists for each of up to three synthetic steps are conveniently associated with building block lists using the first application, CodeGen. The second application Decode allows the user to identify the individual beads picked onto a 96-well plate and the pool number for each bead. The decoding chromatography data for each well is then loaded into the program. The chromatography data is used to identify the tags used in the synthesis. Along with the building block information from ISIS/Host, the building block used in each step of the synthesis can be identified. A third routine, Code-to-Structure, takes the coded library building blocks and creates the connection table in ISIS for each structure found by the decode program. For quality control of encoded library synthesis, the decoded structures on a set of beads is compared to the LC/UV/MS data for the ligand cleaved from the same bead. CAPTURE, a GlaxoSmithKline proprietary application, is used to display and analyze the decoded structures and associated mass spectral data. This application uses simple isotopic composition and electrospray ionization rule sets to predict mass spectra and judge the concordance of a structure- mass spectrum data set. An ancillary program, EIC, is used to extract predicted single ion chromatograms from the full scan LC/MS data.     

Site distribution in resin beads as determined by confocal Raman spectroscopy

Scanning confocal Raman spectroscopy was used to study the distribution of reactive sites within a resin bead used for solid-phase synthesis. The distribution of NH2 groups in aminomethylated polystyrene resin (APS) was determined by doping with varying amounts of 4-cyanobenzoic acid. The extent of loading was determined by both elemental analysis and ninhydrin assays. The spatial distribution of the coupled 4-cyanobenzamide within the bead was determined to an in-plane resolution of 1 microm and depth resolution of about 4 microm, using the strong Raman CN stretching vibrational transition at 2230 cm(-1). Dry and swollen beads were studied and the distribution was found to be essentially uniform throughout the bead in all cases.     

Effect of surface nanotopography on immunoaffinity cell capture in microfluidic devices

Immunoaffinity microfluidic devices have recently become a popular choice to isolate specific cells for many applications. To increase cell capture efficiency, several groups have employed capture beds with nanotopography. However, no systematic study has been performed to quantitatively correlate surface nanopatterns with immunoaffinity cell immobilization. In this work, we controlled substrate topography by depositing close-packed arrays of silica nanobeads with uniform diameters ranging from 100 to 1150 nm onto flat glass. These surfaces were functionalized with a specific antibody and assembled as the base in microfluidic channels, which were then used to capture CD4+ T cells under continuous flow. It is observed that capture efficiency generally increases with nanoparticle size under low flow rate. At higher flow rates, cell capture efficiency becomes increasingly complex; it initially increases with the bead size then gradually decreases. Surprisingly, capture yield plummets atop depositions of some particle diameters. These dips likely stem from dynamic interactions between nanostructures on the substrate and cell membrane as indicated by roughness-insensitive cell capture after glutaraldehyde fixing. This systematic study of surface nanotopography and cell capture efficiency will help optimize the physical properties of microfluidic capture beds for cell isolation from biological fluids.     

From the one-bead-one-compound concept to one-bead-one-reactor

The one-bead-one-compound method gives access to millions of compounds that can be screened directly on the bead. Although characterization techniques are increasingly potent and reliable, problems can still be encountered in deciphering the sequence of the active compound because of sensitiveness or manipulation of the bead. ChemMatrix, a totally PEG-based resin, has resolved the synthesis of peptides of outstanding difficulty. Like other PEG-based resins, it permits on-bead screening because of its compatibility in aqueous media and has the further advantage of having a high loading, comparable to polystyrene resins. ChemMatrix beads previously swelled in water can be nicely divided into four parts that can be characterized using different analytical techniques or just stored for safety or for further testing. The four bead parts show high homogeneity and can thus be considered to be replicas.     

Eine neuartige Methode zur Analyse kombinatorischer Substanzbibliotheken mittels paramagnetischer Reporter

A Novel Method to Identify Chemical Compounds of Combinatorial Libraries by the Use of Paramagnetic Tags An EPR method to identify non‐destructively chemical compounds bound to a single solid‐phase‐synthesis bead for combinatorial chemistry applications is discribed. During synthesis chemical inert paramagnetic substances can be attached in small amounts to a solid‐phase‐synthesis resin for tagging of organic compounds or even reaction steps. The identification of single members of a combinatorial library in short time and high sensitivity can be carried out by using an EPR‐spectrometer.     

Measuring binding of protein to gel-bound ligands using magnetic levitation

This paper describes the use of magnetic levitation (MagLev) to measure the association of proteins and ligands. The method starts with diamagnetic gel beads that are functionalized covalently with small molecules (putative ligands). Binding of protein to the ligands within the bead causes a change in the density of the bead. When these beads are suspended in a paramagnetic aqueous buffer and placed between the poles of two NbFeB magnets with like poles facing, the changes in the density of the bead on binding of protein result in changes in the levitation height of the bead that can be used to quantify the amount of protein bound. This paper uses a reaction-diffusion model to examine the physical principles that determine the values of rate and equilibrium constants measured by this system, using the well-defined model system of carbonic anhydrase and aryl sulfonamides. By tuning the experimental protocol, the method is capable of quantifying either the concentration of protein in a solution, or the binding affinities of a protein to several resin-bound small molecules simultaneously. Since this method requires no electricity and only a single piece of inexpensive equipment, it may find use in situations where portability and low cost are important, such as in bioanalysis in resource-limited settings, point-of-care diagnosis, veterinary medicine, and plant pathology. It still has several practical disadvantages. Most notably, the method requires relatively long assay times and cannot be applied to large proteins (>70 kDa), including antibodies. The design and synthesis of beads with improved characteristics (e.g., larger pore size) has the potential to resolve these problems.     

A new method of immobilizing enzymes by radiopolymerization under low temperature

A new method of immobilizing enzymes by ionizing radiation is described. The mixed aqueous solution of enzyme and polymerizing reagents were quickly frozen at about -70°C then were irradiated with 200 to 500 Krad by⁶⁰Co γ ray. Irradiation was conducted aerobically under the low temperature. The enzyme was entraped in the resulting polymer. As the polymerizing reagent some water soluble polymers having vinyl bonds were also applicable. By this method an immobilized enzyme was prepared in bead, membrane, bag, or tube form having high enzymic activity. When the bead form was to be prepared, the mixture of enzyme and reagents were injected into precooled solvent such as n-hexane, toluene, or petroleum ether. the size of the bead was controlled freely from 10 μm to 1 cm in diameter. The surface of the bead had numerous small holes and the cross section of the bead showed a spongy structure. some acrylates were suitable for the immobilization of enzymes which required the corresponding metal ion as the essential substance. Microorganisms and multienzymes will be immobilized by this technique. This method is inexpensive, quick, simple, and reliable. Immobilized microbial cells can be sterilized by γ irradiation. Invertase was immobilized and the application test was conducted in an enzyme column.     

Measurement of concentration gradients in swelling crosslinked polymer beads

The concentration of solvent in the gel phase of a swelling polystyrene–divinylbenzene copolymer bead has been measured as a function of the radius by optical interference techniques. It is found that an appreciable gradient exists even shortly after the core disappears. Results are presented for two cases: a very lightly crosslinked bead that exhibits negligible birefringence while swelling, and a more tightly crosslinked network in which the birefringence must be considered. It is also found that the refractive index of the unswollen bead is considerably less than that found by extrapolation from the swollen state; this effect is reasonable when the “free volume” in the bulk polymer is considered.     

Acyl hydrazides as peptoid sub-monomers

The use of acyl hydrazides as peptoid sub-monomers is investigated. We demonstrate here that azapeptoids derived entirely from acyl hydrazides can be made conveniently and efficiently using standard peptoid sub-monomer chemistry. Structural studies reveal that the main chain amide bond in these molecules predominantly adopts a trans conformation. A high-quality one bead one compound library of tetramers was made by split and pool synthesis and we found that the identity of the molecule on a single bead could be determined by tandem MALDI mass spectrometry.     

Microfluidic immunomagnetic multi-target sorting--a model for controlling deflection of paramagnetic beads

We describe a microfluidic system that uses a magnetic field to sort paramagnetic beads by deflecting them in the direction normal to the flow. In the experiments we systematically study the dependence of the beads' deflection on bead size and susceptibility, magnet strength, fluid speed and viscosity, and device geometry. We also develop a design parameter that can aid in the design of microfluidic devices for immunomagnetic multi-target sorting.