Progress toward multiplexed sample-to-result detection in low resource settings using microfluidic immunoassay cards

In many low resource settings multiple diseases are endemic. There is a need for appropriate multi-analyte diagnostics capable of differentiating between diseases that cause similar clinical symptoms. The work presented here was part of a larger effort to develop a microfluidic point-of-care system, the DxBox, for sample-to-result differential diagnosis of infections that present with high rapid-onset fever. Here we describe a platform that detects disease-specific antigens and IgM antibodies. The disposable microfluidic cards are based on a flow-through membrane immunoassay carried out on porous nitrocellulose, which provides rapid diffusion for short assay times and a high surface area for visual detection of colored assay spots. Fluid motion and on-card valves were driven by a pneumatic system and we present designs for using pneumatic control to carry out assay functions. Pneumatic actuation, while having the potential advantage of inexpensive and robust hardware, introduced bubbles that interfered with fluidic control and affected assay results. The cards performed all sample preparation steps including plasma filtration from whole blood, sample and reagent aliquoting for the two parallel assays, sample dilution, and IgG removal for the IgM assays. We demonstrated the system for detection of the malarial pfHRPII antigen (spiked) and IgM antibodies to Salmonella Typhi LPS (patient plasma samples). All reagents were stored on card in dry form; only the sample and buffer were required to run the tests. Here we detail the development of this platform and discuss its strengths and weaknesses.     

Long-term storage of bionanodevices by freezing and lyophilization

Successful long-term storage of a "smart dust" device integrating biomolecular motors and complex protein assemblies has been demonstrated using freezing or lyophilization, which implies that fabrication and application can be separated even for complex bionanodevices.     

Structural and magnetic properties of Mg substituted NiCuZn Nano Ferrites

The present paper examines the effect of magnesium substitution on structural and magnetic properties of NiCuZn nano ferrites synthesised by sol-gel method. The prepared samples were characterised by using X-ray Diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Field Emission Scanning Electron Microscopy (FE-SEM) and Vibration sample magnetometer (VSM) techniques. The phase identification, unit cell parameter and crystallite size was determined using XRD analysis. The lattice constant reduced with increasing Mg content. Room temperature saturation magnetisation and coercivity showed reverse trend with increasing Mg content. Curie temperature (T_c) obtained from the thermo magnetic curves increases with Mg concentration. The initial permeability (μ_i) decreased with increasing Mg content. This is due to reduced magnetisation, grain size and increased magneto-crystalline anisotropy constant. Simultaneously, there is an upward shift of domain wall relaxation frequency with increasing Mg content. Also the permeability is observed to be constant up to 30 MHz frequency range showing compositional stability and quality of the material. The prepared samples were suitable for applications in Multilayer Chip Inductors due to their invariable permeability up to 30 MHz frequency and high thermal stability along with low sintering temperature.     

The Tate-Shafarevich Group for Elliptic Curves with Complex Multiplication II

Using Iwasawa theory, we establish some numerical results, and one weak theoretical result, about the enigmatic Tate-Shafarevich group of an elliptic curve defined over the rational field, with complex multiplication. These strengthen results of this kind proven in an earlier paper of ours.     

Enzyme immunoassay for aflatoxin B1-lysine adduct and its validation

A simple procedure was developed for in vitro synthesis and characterization of aflatoxin B1-lysine adduct using aflatoxin B1, N-alpha-acetyl lysine and m-chloroperbenzoic acid (MCPBA). At a molar ratio of 1:16 (aflatoxin B1:N-alpha-cetyl lysine), the recovery of adduct was 62%. Analysis of the adduct by thin-layer chromatography showed a single spot (Rf = 0). Absorption spectra of the adduct showed 2 peaks at 275 and 335 nm. Liquid chromatographic (LC) analysis of the AFB1-lysine adduct showed a relative retention time of 2.1 min. Using the same epoxidation procedure, BSA-AFB1 adduct and ovalbumin-AFB1 adduct were synthesized for production of antibodies and as coating antigen, respectively. Control rat serum, spiked with AFB1-lysine adduct and subjected to LC analysis showed a retention time of 2.1 min, which is similar to that of AFB1-lysine reference standard, synthesized. Further, enzymatically hydrolyzed, control rat serum spiked with BSA-AFB1 adduct showed 2 peaks with retention times of 2.1 and 2.7 min. Based on the LC analysis, recovery of BSA-AFB1 in terms of AFB1-lysine adducts was 67 +/- 5%. The major peak (2.1 min) accounted for 72% of the adduct; the second minor peak (2.7 min) accounted for 28% of the total AFB1-lysine adducts formed. Stability studies on the AFB1-lysine adduct synthesized, indicated that it was stable for 1 month. Antibody capture assay showed an absorbance of 0.9 to 1.0 at a dilution of 1:50,000 when ovalbumin-AFB1 was used as a coating antigen. Indirect competitive ELISA showed 50% displacement (IC50) of the antibodies at a concentration of 13 ng AFB1-lysine, whereas the IC50 for AFB1 was 7 ng. The recovery of AFB1-lysine adduct spiked to control rat serum followed by enzymatic hydrolysis and immunoanalysis (indirect ELISA) was 93 +/- 6%. The enzyme immunoassay was validated by a rodent model, in which the animals were exposed to aflatoxin B1 (20 microg AFB1/kg body mass/day). The level of AFB1-lysine adduct in the rat serum was 27.3 +/- 4.37 microg/mg albumin.     

Exogenous bridging and nonbridging in Cu(II) complexes of Mannich base ligands: Synthesis and physical properties

Preparation of pentadentate ligands L¹, L², L³ and L⁴, where L¹ = 4-chloro-3-methyl-2[(prolin-1-yl)methyl]-6-[N-phenyl piperazin-1-yl)methyl]phenol, L² = 4-ethyl-2-[(prolin-1-yl)methyl]-6-[(N-phenyl piperazin-1-yl)methyl]phenol, L³ = 4-chloro-3-methyl-2-[(prolin-1-yl)methyl]-6-[N-methyl piperazin-1-yl]methyl phenol, L⁴ = 4-methoxy-2-[(prolin-1-yl)methyl]-6-[(N-phenyl piperazin-1-yl)methyl]phenol is described together with that of the corresponding Cu(II) complexes with various bridging motifs like OH, OAc and NO₂. The complexes are characterized by elemental analysis, electrochemical and electron paramagnetic spectral studies. Redox properties of the complexes in acetonitrile are highly quasireversible due to the chemical or/and stereochemical changes subsequent to electron transfer. The complexes show resolved copper hyperfine EPR at room temperature, indicating the presence of weak antiferromagnetic coupling between the copper atoms. Strengths of the antiferromagnetic interactions are in the order NO₂>OAc>OH.     

Tripodal pyrene chromophores for semiconductor sensitization: new footprint design

Large footprint tripodal linkers for metal oxide nanoparticle sensitization, substituted with pyrene as the dye, and three COOR binding groups in para or meta position, were prepared to study the effect of the anchoring group position and of the footprint size on the sensitization processes. Two tripods based on tetraphenylsilane, (1-pyrenyl-4-ethynyl-phenyl)-tris(4-carbomethoxyphenyl-4-ethynyl-phenyl)silane, and (1-pyrenyl-4-ethynyl-phenyl)-tris(4-(4-methoxybenzyloxycarbonyl)phenyl-4-ethynyl-phenyl)silane, decomposed during hydrolysis, while the tetraphenyladamantane derivative, 1-(1-pyrenyl-4-ethynyl-phenyl)-3,5,7-(3-carbomethoxyphenyl-4-ethynyl-phenyl)adamantane, was chemically stable and was readily converted into the corresponding acid and bound to TiO₂ films. The FT-IR-ATR spectrum of 1-(1-pyrenyl-p-ethynyl-phenyl)-3,5,7-(3-carboxyphenyl-4-ethynyl-phenyl)adamantane bound to TiO₂ nanoparticles showed bands characteristic of carboxylate bidentate bonds. The UV-vis absorption and fluorescence emission in THF solution at room temperature were typical of pyrenes substituted with oligophenyleneethynylene linkers.     

Motivic cohomology and unramified cohomology of quadrics

This is the last of a series of three papers where we compute the unramified cohomology of quadrics in degree up to 4. Complete results were obtained in the two previous papers for quadrics of dimension ≤4 and ≥11. Here we deal with the remaining dimensions between 5 and 10. We also prove that the unramified cohomology of Pfister quadrics with divisible coefficients always comes from the ground field, and that the same holds for their unramified Witt rings. We apply these results to real quadrics. For most of the paper we have to assume that the ground field has characteristic 0, because we use Voevodsky’s motivic cohomology. Received August 18, 1999 / final version received December 10, 1999?Published online April 19, 2000     

Multicomponent Efficient Synthesis of New [1,2,4]Triazolo[3,4]thiadiazines

A series of novel triazolo thiadiazines were synthesized by a simple, highly efficient, one‐pot pseudo four component approach involving the condensation of 4‐amino‐5‐hydrazinyl‐4H‐1,2,4‐triazole‐3‐thiol ( 1 ), aromatic aldehydes ( 2 ), and various phenacyl bromides ( 3 ) with good yields. The structures of compounds were confirmed by analytical and spectral (IR, ¹H NMR, ¹³C NMR) data.