Surface modification for enhancing antibody binding on polymer-based microfluidic device for enzyme-linked immunosorbent assay

A novel surface treatment method using poly(ethyleneimine) (PEI), an amine-bearing polymer, was developed to enhance antibody binding on the poly(methyl methacrylate) (PMMA) microfluidic immunoassay device. By treating the PMMA surface of the microchannel on the microfluidic device with PEI, 10 times more active antibodies can be bound to the microchannel surface as compared to those without treatment or treated with the small amine-bearing molecule, hexamethylenediamine (HMD). Consequently, PEI surface modification greatly improved the immunoassay performance of the microfluidic device, making it more sensitive and reliable in the detection of IgG. The improvement can be attributed to the spacer effect as well as the functional amine groups provided by the polymeric PEI molecules. Due to the smaller dimensions (140x125 microm) of the microchannel, the time required for antibody diffusion and adsorption onto the microchannel surface was reduced to only several minutes, which was 10 times faster than the similar process carried out in 96-well plates. The microchip also had a wider detection dynamic range, from 5 to 1000 ng/mL, as compared to that of the microtiter plate (from 2 to 100 ng/mL). With the PEI surface modification, PMMA-based microchips can be effectively used for enzyme linked immunosorbent assays (ELISA) with a similar detection limit, but much less reagent consumption and shorter assay time as compared to the conventional 96-well plate.     

Rapid determination of complex mixtures by dual-column gas chromatography with a novel stationary phase combination and spectrometric detection

Fast GC separations of a broad range of analytes are demonstrated using a capillary column coated with a novel immobilized ionic liquid (IIL) stationary phase. Both completely cross-linked and partially cross-linked columns were evaluated, yielding approximately 1600 and approximately 2000 theoretical plates per meter, respectively. Enhanced separation is demonstrated using a dual-column ensemble comprised of an IIL column, a commercially coated Rtx-1 column, and a pneumatic valve connecting the inlet to the junction point between the two columns. Enhanced separation of 20 components, with two sets of co-eluting peaks is shown in approximately 150 s, while sacrificing only a length of time equivalent to the sum of the stop flow pulses, or about 15.5 s. A novel application of a band trajectory model that shows band position as a function of analysis time as analytes move through the column ensemble is employed to determine pulse application times. The model predicts component retention times within a few seconds. Another method of selectivity enhancement of the IIL stationary phase-coated columns is demonstrated using a differential mobility spectrometer (DMS) that provides a second dimension separation based on ion mobility in a high-frequency electrical field. The DMS is able to separate all but one set of co-eluting components from the IIL column. The separation of 13 components found in the headspace above U.S. currency is demonstrated using the IIL column in a dual-column ensemble as well as with the DMS.     

Evaluation of thiol‐ene click chemistry in functionalized polysiloxanes

Polysiloxanes are commonly used in a myriad of applications, and the “click” nature of the thiol‐ene reaction is well suited for introducing alternative functionalities or for crosslinking these ubiquitous polymers. As such, understanding of the thiol‐ene reaction in the presence of silicones is valuable and would lead to enhanced methodologies for modification and crosslinking. Here, the thiol‐ene reaction kinetics were investigated in functionalized oligosiloxanes having varying degrees of thiol functionalization (SH), π–π interactions (from diphenyls, DP), and ene types (C?C). In the ene‐functionalized oligomers, π–π interactions were controlled through the use of dioctyl repeats (DO). The polymerization rate and rate‐limiting steps were determined for all systems containing an allyl‐functionalized oligomer, and rates ranging from 0.10 to 0.54 mol L^?1 min^?1 were seen. The rate‐limiting step varied with the oligomer composition; examples of rate‐limited propagation (5:3:2 C?C:DP:DO/1:1 SH:DP) or chain transfer (5:3:2 C?C:DP:DO/3:1 SH:DP) were found in addition to cases with similar reaction rate constants (5:2:3 C?C:DP:DO/1:1 SH:DP). None of the siloxanes were found to exhibit autoacceleration despite their relatively high viscosities. Instead, the allyl‐, vinyl‐, and acrylate‐functionalized siloxanes were all found to undergo unimolecular termination based on their high α scaling values (0.98, 0.95, and 0.82, respectively) in the relation R_p ∝ R_i^α. © 2013 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

The effect of molecular weight on the separation of semiconducting single‐walled carbon nanotubes using poly(2,7‐carbazole)s

The use of selective interactions between conjugated polymers and single‐walled carbon nanotubes has emerged as a promising method for the separation of nanotubes by electronic type. Although much attention has been devoted to investigating polyfluorenes and their ability to disperse semiconducting carbon nanotubes under specific conditions, other polymer families, such as poly(2,7‐carbazole)s, have been relatively overlooked. Poly(2,7‐carbazole)s have been shown to also preferentially interact with semiconducting carbon nanotubes, however a detailed investigation of polymer parameters, such as molecular weight, has not been performed. We have prepared seven different molecular weights of a poly(2,7‐carbazole), from short chain oligomers to high molecular weight polymers, and have investigated their effectiveness at dispersing semiconducting single‐walled carbon nanotubes. Although all polymer chain lengths were able to efficiently exfoliate carbon nanotube bundles using a mild dispersion protocol, only polymers above a certain threshold molecular weight (M_n ～ 27 kDa) were found to exhibit complete selectivity for semiconducting nanotubes, with no observable signals from metallic species. Additionally, we found the quality of separation to be strongly dependent on the ratio of polymer to carbon nanotube. Contrary to previous reports, we have found that an excess of poly(2,7‐carbazole) leads to incomplete removal of metallic carbon nanotubes. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2015 , 53, 2510–2516     

Beyond microstructures: Using the Kerr Effect to characterize the macrostructures of synthetic polymers

The macrostructures of synthetic polymers are essentially the complete molecular chain architectures, including the types and amounts of constituent short‐range microstructures, such as the regio‐ and stereosequences of the inserted monomers, the amounts and sequences of monomers found in co‐, ter‐, and tetra‐polymers, branching, inadvertent, and otherwise, etc. Currently, the best method for characterizing polymer microstructures uses high field, high resolution ¹³C‐nuclear magnetic resonance (NMR) spectroscopy observed in solution. However, even ¹³C‐NMR is incapable of determining the locations or positions of resident polymer microstructures, which are required to elucidate their complete macrostructures. The sequences of amino acid residues in proteins, or their primary structures, cannot be characterized by NMR or other short‐range spectroscopic methods, but only by decoding the DNA used in their syntheses or, if available, X‐ray analysis of their single crystals. Similarly, there are currently no experimental means to determine the sequences or locations of constituent microstructures along the chains of synthetic macromolecules. Thus, we are presently unable to determine their macrostructures. As protein tertiary and quaternary structures and their resulting ultimate functions are determined by their primary sequence of amino acids, so too are the behaviors and properties of synthetic polymers critically dependent on their macrostructures. We seek to raise the consciousness of both synthetic and physical polymer scientists and engineers to the importance of characterizing polymer macrostructures when attempting to develop structure–property relations. To help achieve this task, we suggest using the electrical birefringence or Kerr effects observed in their dilute solutions. The molar Kerr constants of polymer solutes contributing to the birefringence of their solutions, under the application of a strong electric field, are highly sensitive to both the types and locations of their constituent microstructures. As a consequence, we may begin to characterize the macrostructures of synthetic polymers by means of the Kerr effect. To simplify implementation of the Kerr effect to characterize polymer macrostructures, we suggest that NMR first be used to determine the types and amounts of constituent microstructures present. Subsequent comparison of observed Kerr effects with those predicted for different microstructural locations along the polymer chains can then be used to identify the most likely macrostructures. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2015 , 53, 155–166     

Isolation of tumor cells using size and deformation

The isolation and analysis of circulating tumor cells (CTCs) from blood are the subject of intense research. Although tests to detect metastasis on a molecular level are available, progress has been hampered by a lack of tumor-specific markers and predictable DNA abnormalities. The main challenge in this endeavor is the small number of available cells of interest, 1–2 per mL in whole blood. We have designed a micromachined device to fractionate whole blood using physical means to enrich for and/or isolate rare cells from peripheral circulation. It has arrays of four successively narrower channels, each consisting of a two-dimensional array of columns. Current devices have channels ranging in width from 20 to 5 μm, and in depth from 20 to 5 μm. Several optimizations resulting in the fabrication of a total of 10 derivative devices have been carried out; only two types are used in this study. Both have increasingly narrower gap widths between the columns along the flow axis with 20, 15, 10, and 5 μm spacing all on one device. The first 20 μm wide segment disperses the cell suspension and creates an evenly distributed flow over the entire device, whereas the others were designed to retain increasingly smaller cells. The channel depth is constant across the entire device, the first type was 10 μm deep and the second type is 20 μm deep. When cells from each of eight tumor cell lines were loaded into the device, all cancerous cells were isolated. In mixing experiments using human whole blood, we were able to fractionate cancer cells without interference from the blood cells. Additionally, either intact cells, or DNA, could be extracted for molecular analysis. The ultimate goal of this work is to characterize the cells on the molecular level to provide non-invasive methods to monitor patients, stage disease, and assess treatment efficacy. Furthermore, this work will use gene expression profiles to gain insights into metastasis.     

Nickel‐Catalyzed Synthesis of Dialkyl Ketones from the Coupling of N‐Alkyl Pyridinium Salts with Activated Carboxylic Acids

While ketones are among the most versatile functional groups, their synthesis remains reliant upon reactive and low‐abundance starting materials. In contrast, amide formation is the most‐used bond‐construction method in medicinal chemistry because the chemistry is reliable and draws upon large and diverse substrate pools. A new method for the synthesis of ketones is presented here that draws from the same substrates used for amide bond synthesis: amines and carboxylic acids. A nickel terpyridine catalyst couples N‐alkyl pyridinium salts with in situ formed carboxylic acid fluorides or 2‐pyridyl esters under reducing conditions (Mn metal). The reaction has a broad scope, as demonstrated by the synthesis of 35 different ketones bearing a wide variety of functional groups with an average yield of 60±16 %. This approach is capable of coupling diverse substrates, including pharmaceutical intermediates, to rapidly form complex ketones.     

Transient ordering in a quasi-two-dimensional liquid near freezing

We report the results of a theoretical study of locally ordered fluctuations in a quasi-two-dimensional colloid fluid. The fluctuations in the equilibrium state are monitored by the aperture cross-correlation function of radiation scattered by the fluid, as calculated from molecular dynamics simulations of near hard spheres with diameter sigma confined between smooth hard walls. These locally ordered fluctuations are transient; their decay can be monitored as a function of the time between the cross-correlated scattered radiation signals, but only the single-time cross-correlated signals are discussed in this paper. Systems with thicknesses less than two hard sphere diameters were studied. For wall separation H in the range 1 sigma/=1.57 sigma, hexagonal fluctuations persist in the dense liquid up to H=1.75 sigma, and fluctuations with square ordered symmetry, that of the solid to which the liquid freezes, only emerge at densities approximately 2% below freezing. For H=1.8 sigma and 1.85 sigma, hexagonal ordered flucuations are no longer found, and the square ordered fluctuations dominate the dense liquid region as the system freezes into a two layer square solid. For H=1.9 sigma and 1.95 sigma, where the liquid freezes into a two layer hexagonal solid, both square and hexagonal ordered fluctuations are observed. At lower densities, the ordered fluctuations only exhibit square symmetry. Hexagonal ordered fluctuations appear at densities approximately 7% below freezing and become more dominant as the density is increased, but the square ordered fluctuations persist until the system is converted into the solid.     

Optimal preparation methods for automated matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry profiling of low molecular weight proteins and peptides

Mass spectrometry (MS) profiling of the proteome and peptidome for disease‐associated patterns is a new concept in clinical diagnostics. The technique, however, is highly sensitive to external sources of variation leading to potentially unacceptable numbers of false positive and false negative results. Before MS profiling can be confidently implemented in a medical setting, standard experimental methods must be developed that minimize technical variance. Past studies of variance have focused largely on pre‐analytical variation (i.e., sample collection, handling, etc.). Here, we examined how factors at the analytical stage including the matrix and solid‐phase extraction influence MS profiling. Firstly, a standard peptide/protein sample was measured automatically by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS across five consecutive days using two different preparation methods, dried droplet and sample/matrix, of four types of matrix: α‐cyano‐4‐hydroxycinnamic acid (HCCA), sinapinic acid (SA), 2,5‐dihydroxybenzoic acid (DHB) and 2,5‐dihydroxyacetophenone (DHAP). The results indicated that the matrix preparation greatly influenced a number of key parameters of the spectra including repeatability (within‐day variability), reproducibility (inter‐day variability), resolution, signal strength, background intensity and detectability. Secondly, an investigation into the variance associated with C8 magnetic bead extraction of the standard sample prior to automated MS profiling demonstrated that the process did not adversely affect these same parameters. In fact, the spectra were generally more robust following extraction. Thirdly, the best performing matrix preparations were evaluated using C8 magnetic bead extracted human plasma. We conclude that the DHAP prepared according to the dried‐droplet method is the most appropriate matrix to use when performing automated MS profiling. Copyright © 2009 John Wiley & Sons, Ltd.