Using light to bioactivate surfaces: A new way of creating oriented, active immunobiosensors

Ultraviolet light can be used to immobilize biomolecules onto thiol reactive surfaces in order to, e.g., make biosensors. The mechanism involves light-induced formation of free, reactive thiol groups in disulphide containing molecules. This technology allows for the creation of arrays of biomolecules with a high degree of reproducibility, circumventing the need for often expensive nano/micro-dispensing technologies. The ultimate size of the immobilized spots is defined by the focal area of the UV beam. Light-induced immobilization has the added benefit that the immobilized molecules will be spatially oriented and covalently bound to the surface. In this paper, we demonstrate the utility of a sensor array created with the new sensor technology when integrated into a microfluidic system. Protein arrays made using light-induced immobilization showed successful antigen/antibody binding in a flow cell allowing the visualisation of real time binding and enzyme activity. This new technology is ideal for the creation of protein/DNA microarrays, can replace present micro-dispensing arraying technologies and is ideal as a molecular imprinting technology.     

AFM study of BSA adlayers on Au stripes

Thin narrow Au stripes suitable for propagating long-range surface plasmon-polaritons were deposited by evaporation and lift-off on a thermal oxide layer on a silicon substrate, and modified by direct adsorption of bovine serum albumin (BSA). Atomic force microscopy (AFM) measurements reveal that BSA adsorbs onto the Au stripes from phosphate buffer solutions forming an adlayer having an average thickness of about 2 nm (surface mass density of about 2 ng/mm²). Comparisons with a simple adsorption model suggest the side-on adsorption of a single monolayer of BSA followed by denaturation and flattening. The BSA-coated stripes have an increased surface roughness compared to a virgin stripe.     

Quantification of ATRP initiator density on polymer latex particles by fluorescence labeling technique using copper‐catalyzed azide‐alkyne cycloaddition

We developed a novel fluorescence labeling technique for quantification of surface densities of atom transfer radical polymerization (ATRP) initiators on polymer particles. The cationic P(St‐CPEM‐C₄DMAEMA) and anionic P(St‐CPEM) polymer latex particles carrying ATRP‐initiating chlorine groups were prepared by emulsifier‐free emulsion polymerization of styrene (St), 2‐(2‐chloropropionyloxy)ethyl methacrylate (CPEM), and N‐n‐butyl‐N,N‐dimethyl‐N‐(2‐methacryloyloxy)ethylammonium bromide (C₄DMAEMA). ATRP initiators on the surface of polymer particles were converted into azide groups by sodium azide, followed by fluorescent labeling with 5‐(N,N‐dimethylamino)‐N′‐(prop‐2‐yn‐1‐yl)naphthalene‐1‐sulfonamide (Dansyl‐alkyne) by copper‐catalyzed azide‐alkyne cycloaddition (CuAAC). The reaction time required for both azidation of ATRP‐initiating groups and successive fluorescence labeling of azide groups with Dansyl‐alkyne by CuAAC were investigated in detail by FTIR and fluorescence spectral measurement, respectively. The ATRP initiator densities on the cationic P(St‐CPEM‐C₄DMAEMA) and anionic P(St‐CPEM) particle surfaces were estimated to be 0.21 and 0.15 molecules nm^?2, respectively, which gave close agreement with values previously determined by a conductometric titration method. The fluorescence labeling through click chemistry proposed herein is a versatile technique to quantify the surface ATRP initiator density both on anionic and cationic polymer particles. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013 , 51, 4042–4051     

An ultra‐high temperature flow‐through capillary device for bacterial spore lysis

Rapid and specific characterization of bacterial endospores is dependent on the ability to rupture the cell wall to enable analysis of the intracellular components. In particular, bacterial spores from the bacillus genus are inherently robust and very difficult to lyze or solubilize. Standard protocols for spore inactivation include chemical treatment, sonication, pressure, and thermal lysis. Although these protocols are effective for the inactivation of these agents, they are less well suited for sample preparation for analysis using proteomic and genomic approaches. To overcome this difficulty, we have designed a simple capillary device to perform thermal lysis of bacterial spores. Using this device, we were able to super heat (195°C) an ethylene glycol lysis buffer to perform rapid flow‐through rupture and solubilization of bacterial endospores. We demonstrated that the lysates from this preparation method are compatible with CGE as well as DNA amplification analysis. We further demonstrated the flow‐through lysing device could be directly coupled to a miniaturized electrophoresis instrument for integrated sample preparation and analysis. In this arrangement, we were enabled to perform sample lysis, fluorescent dye labeling, and protein electrophoresis analysis of bacterial spores in less than 10 min. The described sample preparation device is rapid, simple, inexpensive, and easily integratable with various microfluidic devices.     

Development of a high-sensitivity chromatographic separation system for pyridylaminated aldopentoses and aldohexoses

A rare sugar is considered to be a monosaccharide rarely found in nature. To investigate their natural distribution and biological roles, a robust analytical system must be used to isolate, identify, and quantify them. Herein, we report the development of such a system that can specifically quantify and chromatographically separate four aldopentoses and eight aldohexoses tagged with 2-aminopyridine. Purified monosaccharides derivatized with a pyridylamino moiety (PA–monosaccharides) are first chromatographed over a high-performance anion-exchange resin. But, because two of the PA–aldohexoses used in this study, PA–talose and PA–idose, co-elute with the common saccharides, PA–glucose and PA–mannose, respectively, a second chromatographic step, reversed-phase high-performance liquid chromatography, is used to completely separate them. Thus, as shown by the results of this study, chromatographic separation of PA–monosaccharides is achievable and provides a quantitative measurement of common and rare isomeric aldopentoses and aldohexoses.     

Transfer Thermodynamics of Protein in Denaturing and Stabilizing Media

Free energies of transfer (ΔG_t) of RibonucleaseA (RNaseA) from water to aqueous solutions of urea (4 M, 6 M and 8 M), a protein denaturing solvent as well as ΔG_t of RibonucleaseA, β‐Lactoglobulin, α‐Chymotripsin and ChymotrypsinogenA from water to aqueous glycerol (10%, 20%, 30% and 40%), a protein stabilizing solvent has been dissected into cavity term [ΔG_t(cav)] and interaction term [ΔG_t(int)]. The interaction free energy includes all types of interactions like hard‐soft, hydrogen bonding, electrostatic, etc. The cavity forming free energies have been calculated using the standard version of scaled particle theory (SPT) with well‐reported SPT parameters. It has been found that transfer free energies of cavity terms ΔG_t(cav) for native protein from water to urea‐water and water to aqueous glycerol follow almost opposite trends. This primarily indicates there may be some correlation between cavity creation energies and protein denaturing and stabilizing ability of a solvent. The results are in agreement with those obtained from preferential binding coefficient studies in these media.     

Relation of structure to performance characteristics of monolithic and perfusive stationary phases

Commercially available polymer-based monolithic and perfusive stationary phases were evaluated for their applicability in chromatography of biologics. Information on bed geometry, including that from electron microscopy (EM), was used to interpret and predict accessible volumes, binding capacities, and pressure drops. For preparative purification of biologics up to at least 7 nm in diameter, monoliths and perfusive resins are inferior to conventional stationary phases due to their low binding capacities (20–30 g/L for BSA). For larger biologics, up to several hundred nanometers in diameter, calculations from EM images predict a potential increase in binding capacity to nearly 100 g/L. The accessible volume for adenovirus calculated from the EM images matched the experimental value. While the pores of perfusive resins are essentially inaccessible to adenovirus under binding conditions, under non-adsorbing conditions the accessible intrabead porosity is almost as large as the interbead porosity. Modeling of breakthrough curves showed that the experimentally observed slow approach to full saturation can be explained by the distribution of pore sizes.     

On friendly index sets of 2-regular graphs

Let G be a graph with vertex set V and edge set E, and let A be an abelian group. A labeling f:V→A induces an edge labeling f^∗:E→A defined by f^∗(xy)=f(x)+f(y). For i∈A, let v_f(i)=card{v∈V:f(v)=i} and e_f(i)=card{e∈E:f^∗(e)=i}. A labeling f is said to be A-friendly if |v_f(i)−v_f(j)|≤1 for all (i,j)∈A×A, and A-cordial if we also have |e_f(i)−e_f(j)|≤1 for all (i,j)∈A×A. When A=Z₂, the friendly index set of the graph G is defined as {|e_f(1)−e_f(0)|:the vertex labelingf is Z₂-friendly}. In this paper we completely determine the friendly index sets of 2-regular graphs. In particular, we show that a 2-regular graph of order n is cordial if and only if n?2 (mod 4).     

Strong edge-magic graphs of maximum size

An edge-magic total labeling on G is a one-to-one map λ from V(G)∪E(G) onto the integers 1,2,…,|V(G)∪E(G)| with the property that, given any edge (x,y), λ(x)+λ(x,y)+λ(y)=k for some constant k. The labeling is strong if all the smallest labels are assigned to the vertices. Enomoto et al. proved that a graph admitting a strong labeling can have at most 2|V(G)|-3 edges. In this paper we study graphs of this maximum size.