Label-Free Impedance Biosensors: Opportunities and Challenges

Impedance biosensors are a class of electrical biosensors that show promise for point-of-care and other applications due to low cost, ease of miniaturization, and label-free operation. Unlabeled DNA and protein targets can be detected by monitoring changes in surface impedance when a target molecule binds to an immobilized probe. The affinity capture step leads to challenges shared by all label-free affinity biosensors; these challenges are discussed along with others unique to impedance readout. Various possible mechanisms for impedance change upon target binding are discussed. We critically summarize accomplishments of past label-free impedance biosensors and identify areas for future research.     

Vacancy ion-exclusion chromatography of carboxylic acids on a weakly acidic cation-exchange resin

In this preliminary study, a new approach to ion-exclusion chromatography is proposed to overcome the relatively poor conductivity detection response which occurs in ion-exclusion chromatography when acids are added to the eluent in order to improve peak shape. This approach, termed vacancy ion-exclusion chromatography, requires the sample to be used as eluent and a sample of water to be injected onto a weakly acidic cation-exchange column (TSKgel OApak-A). Vacancy peaks for each of the analytes appear at the retention times of these analytes. Highly sensitive conductivity detection is possible and sharp, well-shaped peaks are produced, leading to efficient separations. Retention times were found to be affected by the concentration of the analytes in the eluent, and also by the presence of an organic modifier such as methanol in the eluent. Detection limits for oxalic, formic, acetic, propionic, butyric and valeric acids were 0.1, 0.2, 0.3, 0.3, 0.4 and 0.5 microM, respectively, and linear ranges for some acids extended over two orders of magnitude. Precision values for retention times were 0.21% and for peak areas were <1.90%. The vacancy ion-exclusion chromatography method was found to give detection responses four to 10 times higher than conventional ion-exclusion chromatography using sulfuric acid eluent and two to five times higher than conventional ion-exclusion chromatography using benzoic acid eluent.     

Solid-Phase Synthesis of Phenylacetylene Oligomers Utilizing a Novel 3-Propyl-3-(benzyl-supported) Triazene Linkage

Sequence-specific phenylacetylene oligomers consisting of functionalized monomers (hexyl benzoate, hexyl phenyl ether, benzonitrile, and tert-butylphenyl) are synthesized in gram quantities using solid-phase methods. Growing oligomers are attached to a divinylbenzene cross-linked polystyrene support by the 1-aryl-3-propyl-3-(benzyl-supported) triazene moiety. This linkage is obtained by reaction of arenediazonium tetrafluoroborate salts with a n-propylamino-modified Merrifield resin. Condensation strategies are described, producing oligomers with higher yields and simplified procedures compared to solution-phase methods. Terminal acetylene is protected with a trimethylsilyl group. After deprotection of the resin-bound terminal acetylene, an aryl iodide monomer or an aryl iodide-terminated oligomer is coupled to the supported oligomer using a palladium(0) catalyst. The cycle can be repeated to produce sequence-specific oligomers of varying length and functionality. The resulting oligomers are liberated from the polymer support by cleavage of the 1-aryl-3-propyl-3-(benzyl-supported) triazene group by reaction with iodomethane producing an aryl iodide.     

Analytical investigation of the properties and uses of a new hydrophobic molecular sieve

Distribution coefficients and capacities have been determined for many different organic compounds on a new molecular sieve called silicalite, which does not adsorb water but does adsorb small organic molecules with diameters up to 6 A, from both liquid and gaseous streams. The characteristics of silicalite have been examined closely and new applications for it are briefly described.     

Heat shock protein 27 interacts with vimentin and prevents insolubilization of vimentin subunits induced by cadmium

Vimentin is an intermediate filament that regulates cell attachment and subcellular organization. In this study, vimentin filaments were morphologically altered, and its soluble subunits were rapidly reduced via cadmium chloride treatment. Cadmium chloride stimulated three major mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and led apoptotic pathway via caspase-9 and caspase-3 activations. In order to determine whether MAPKs were involved in this cadmium-induced soluble vimentin disappearance, we applied MAPK-specific inhibitors (PD98059, SP600125, SB203580). These inhibitors did not abolish the cadmium-induced soluble vimentin disappearance. Caspase and proteosome degradation pathway were also not involved in soluble vimentin disappearance. When we observed vimentin levels in soluble and insoluble fractions, soluble vimentin subunits shifted to an insoluble fraction. As we discovered that heat-shock protein 27 (HSP27) was colocalized and physically associated with vimentin in unstressed cells, the roles of HSP27 with regard to vimentin were assessed. HSP27-overexpressing cells prevented morphological alterations of the vimentin filaments, as well as reductions of soluble vimentin, in the cadmium-treated cells. Moreover, HSP27 antisense oligonucleotide augmented these cadmium-induced changes in vimentin. These findings indicate that HSP27 prevents disruption of the vimentin intermediate filament networks and soluble vimentin disappearance, by virtue of its physical interaction with vimentin in cadmium-treated SK-N-SH cells.     

Stereoselective synthesis of optically active bicyclic beta-lactam carboxylic acids that target pilus biogenesis in pathogenic bacteria

Optically active bicyclic beta-lactams were synthesized, starting from 2-H-delta 2-thiazolines and Meldrum's acid derivatives. Several methods to accomplish an ester hydrolysis without damaging the beta-lactam framework were investigated. A rapid CsOH saponification of the beta-lactam methyl esters was developed and protonation of the Cs-carboxylates by Amberlite (IR-120 H+) afforded a series of bicyclic beta-lactam carboxylic acids. Moreover, a convenient method for the synthesis of 2-H-delta 2-thiazolinecarboxylic acid methyl ester 2 was developed. Bicyclic beta-lactam carboxylic acids 7a-g and aldehydes 4a-d were screened for their affinity to the bacterial periplasmic chaperone PapD using a surface plasmon resonance technique. beta-Lactams substituted with large acyl substitutents showed better binding to the chaperone than the native C-terminal peptide PapG 8, demonstrating that bicyclic beta-lactams constitute a new class of potential bacterial chaperone inhibitors.     

Coulometric titration of acids in non-aqueous solvents

Coulometric titrations of mineral acids, sulphonic acids, carboxylic acids, enols, imides and phenols have been carried out in t-butanol or in acetone with electrically generated tetrabutylammonium hydroxide. Either a potentiometric titration or a visual indicator end-point may be used. The amount of acid titrated ranges from 10 to 60 muequiv, and the precision and accuracy of the method are excellent.     

6-Oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl)fluorescein as a new fluorescent labeling reagent for aliphatic amines in environmental and food samples using high-performance liquid chromatography

6-Oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl)fluorescein (SAMF), a new fluorescein-based amine-reactive fluorescent probe was well designed, synthesized and used as a pre-column derivatizing reagent for the determination of aliphatic amines in HPLC. It exhibited relatively pH-independent fluorescence (pH 4-9) and excellent photostability. The derivatization was performed at room temperature in 6min. On a C18 column, the derivatives of SAMF with eight aliphatic amines were baseline separated in 28 min with a mobile phase of methanol-water (57:43, v/v) containing 10 mmol l(-1) pH 5.0, H3Cit3-NaOH buffer. With fluorescent detection at lambda(ex)/lambda(em) = 484/516 nm, the detection limit could reach 2-320 fmol (signal-to-noise = 3), which was equivalent to or better than the detection limits obtained from other analytical methods of aliphatic amines. The proposed method has been applied to the determination of the aliphatic amines in environmental and food samples such as lake water, red wine, white wine, and cheese with satisfying recoveries varying from 95 to 106%.     

Tris(2-ethylhexyl)phosphate as an extractant for trivalent gallium, indium and thallium

A systematic study of solvent extraction behaviour of gallium(III), indium(III) and thallium(III) with tris(2-ethylhexyl)phosphate from salicylate media has been undertaken and a scheme for their separation is proposed. The optimum extraction conditions are evaluated and described. The method is applicable to the analysis of standard aluminium and aluminium alloys samples. The results obtained are reproducible and accurate.     

Fragmentation of electrospray-produced oligodeoxynucleotide ions adducted to metal ions

In this article, we describe the unique fragmentations of oligodeoxynucleotides (ODNs) whose phosphate groups are completely depleted of protons and replaced with metal ions. The production of the ubiquitous [a(n) - base] ions still occurs, but no longer by transfer of an acidic phosphate proton to an adjoining 3' base. Nor is the extent of the reaction determined by the proton affinity of that base. Rather, the reaction now occurs via a cleavage 3' to both pyrimidines and purines; cleavage 3' to pyrimidine is more favorable than that 3' to purine. We also demonstrate that an ODN is more stable in the gas phase when its phosphate groups are bound to metal ions than when its phosphate groups are attached to hydrogens. This study also provides further evidence for the ODN fragmentation mechanism that involves H transfer to a nucleobase. To establish the structural utility of this new fragmentation, we applied it to distinguishing small ODNs containing a photomodified cis,syn-cyclobutane pyrimidine dimer from the parent ODNs, a system that cannot be distinguished by collisional activation of precursor species that do not contain metal ions.