Ganglioside-liposome immunoassay for the ultrasensitive detection of cholera toxin

An extremely sensitive bioassay has been developed for cholera toxin (CT) detection, using ganglioside-incorporated liposomes. Cholera is a diarrheal disease, often associated with water or seafood contamination. Ganglioside GM1 was used to prepare the liposomes by spontaneous insertion into the phospholipid bilayer. CT recognition and signal generation is based on the strong and specific interaction between GM1 and CT. In a sandwich immunoassay, CT was detected as a colored band on the nitrocellulose membrane strip, where CT bound to GM1-liposomes can be captured by immobilized antibodies. The intensity of the band could be visually estimated or measured by densitometry, using computer software. The limit of detection (LOD) of CT in the assay system was found to be 10 fg/mL which is equivalent to 8 zmol in the 70-microL sample. The assay was also tested with water samples spiked with CT, providing a LOD of 0.1-30 pg/mL, which is much better than previously reported limits of detection from other assays. The assay could be completed within 20 min. These results demonstrate that the bioassay developed for CT is rapid and ultrasensitive, suggesting the possibility for detecting CT, simply and reliably, in field screening.     

Multifunctional gold-silica nanostructures for ultrasensitive electrochemical immunoassay of streptomycin residues

A facile and simple electrochemical immunoassay for ultrasensitive determination of streptomycin residues (STR) in food was designed by using nanogold-assembled mesoporous silica (GMSNs) as bionanolabels on a three-dimensional redox-active organosilica-functionalized sensing interface. To construct such a sensing interface, we initially synthesized organosilica colloids by using wet chemical method, and then utilized the prepared colloidal organosilica nanocomposites for the immobilization of monoclonal anti-STR antibodies on a glassy carbon electrode based on a sol-gel method. The bionanolabels were prepared based on coimmobilization of horseradish peroxidase (HRP) and STR-bovine serum albumin conjugates (STR-BSA) on the GMSNs. With a competitive-type immunoassay format, the assay toward STR analyte was carried out in pH 5.5 acetate acid buffer (ABS) by using redox-active organosilica nanocomposites as electron mediators, biofunctionalized GMSNs as traces, and hydrogen peroxide (H(2)O(2)) as enzyme substrate. Under optimal conditions, the reduction current of the electrochemical immunosensor decreased with the increase in STR level in the sample, and displayed a wide dynamic range of 0.05-50 ng mL(-1) with a low detection limit (LOD) of 5 pg mL(-1) at 3s(B). Intra- and interassay coefficients of variation were less than 8.7 and 9.3% for STR detection, respectively. In addition, the methodology was validated with STR spiked samples including honey, milk, kidney, and muscle, receiving a good correspondence with the results obtained from high-performance liquid chromatography (HPLC).     

One-pot polymerase chain reaction with gold nanoparticles for rapid and ultrasensitive DNA detection

We report a novel method for rapid, colorimetric detection of a specific deoxyribonucleic acid (DNA) sequence by carrying out a polymerase chain reaction in the presence of gold nanoparticles functionalized with two primers. Extension of the primers when the target DNA is present as a template during the polymerase chain reaction process affords the complementary sequences on the gold nanoparticle surfaces and results in the formation of gold nanoparticle aggregates with a concomitant color change from red to pinkish/purple. This method provides a convenient and straightforward solution for ultrasensitive DNA detection without any further post-treatment of the polymerase chain reaction products being necessary, and is a promising tool for rapid disease diagnostics and gene sequencing.     

Nanomechanical sensor for rapid and ultrasensitive detection of tumor markers in serum using nanobody

Early cancer diagnosis requires ultrasensitive detection of tumor markers in blood.To this end,we develop a novel microcantilever immunosensor using nanobodies(Nbs)as receptors.As the smallest antibody(Ab)entity comprising an intact antigen-binding site,Nbs achieve dense receptor layers and short distances between antigen-binding regions and sensor surfaces,which significantly elevate the generation and transmission of surface stress.Owing to the inherent thiol group at the C-terminus,Nbs are covalently immobilized on microcantilever surfaces in directed orientation via one-step reaction,which further enhances the stress generation.For microcantilever-based nanomechanical sensor,these advantages dramatically increase the sensor sensitivity.Thus,Nb-functionalized microcantilevers can detect picomolar concentrations of tumor markers with three orders of magnitude higher sensitivity,when compared with conventional Ab-functionalized microcantilevers.This proof-of-concept study demonstrates an ultrasensitive,label-free,rapid,and low-cost method for tumor marker detection.Moreover,interestingly,we find Nb inactivation on sensor interfaces when using macromolecule blocking reagents.The adsorption-induced inactivation is presumably caused by the change of interfacial properties,due to binding site occlusion upon complex coimmobilization formations.Our findings are generalized to any coimmobilization methodology for Nbs and,thus,for the construction of high-performance immuno-surfaces.     

Development of an aggregation-based immunoassay for anti-protein A using gold nanoparticles

A unique, sensitive, and highly specific immunoassay system for antibodies using gold nanoparticles has been developed. The assay is based on the aggregation of gold nanoparticles that are coated with protein antigens in the presence of their corresponding antibodies. The aggregation of the gold nanoparticles results in an absorption change at 620 nm that is monitored using an absorption plate reader. To demonstrate the analytical capabilities of the new technique, monodispersed protein A-coated gold particles, averaging 10 nm in diameter, were used to determine the level of anti-protein A in serum samples. The effects of the pH, the temperature, and the concentration of protein A-coated gold nanoparticles on the sensitivity of the assay were investigated using transmission electron microscopy (TEM) and UV/vis absorption spectroscopy. A dynamic range of 2 orders of magnitude and a limit of detection of 1 microg/mL of anti-protein A were observed. The new technique could be used for fast, high-throughput screening of antibodies in clinical diagnostic applications.     

Development and evaluation of an immunoassay for biological monitoring chlorophenols in urine as potential indicators of occupational exposure.

Trichlorophenols (TCP) eliminated by the urine can be considered as potential biomarkers of exposure of many chemicals (chlorophenols, chlorophenoxy acid herbicides, prochloraz, lindane, hexachlorobenzene, etc). High-throughput screening methods are necessary to carry out efficient monitoring programs that may help to prevent certain occupational health diseases. For this purpose, an indirect enzyme-linked immunosorbent assay (ELISA) for 2,4,6-trichlorophenol detection has been developed using polyclonal antisera raised against 3-(3-hydroxy-2,4,6-trichlorophenyl)propanoic acid (hapten 5) covalently coupled by the mixed anhydride (MA) method to keyhole limpet hemocyanin (KLH). The indirect ELISA uses a heterologous coating antigen prepared by conjugation of 3-(2-hydroxy-3,6-dichlorophenyl)propanoic acid (hapten 4) to bovine serum albumin (BSA) using the active ester (AE) method. The optimum hapten density for the coating antigen was found to be 3 mol of hapten/mol of protein. The assay shows a limit of detection of 0.245 +/- 0.116 microg L(-1), and it is performed on 96-well microtiter plates in about 1.5 h. The ELISA reported recognizes on a much less extent other chlorinated phenols, such as 2,3,4,6-tetrachlorophenol (2,3,4,6-TtCP, 21%), 2,4,5-TCP (12%) and 2,3,5-TCP (15%); however, brominated phenols (BP) are even more recognized than the corresponding chlorinated analogues (ex. 2,4,6-TBP, 710%; 2,4-DBP, 119%). With the aim of finding an explanation for this behavior, theoretical calculations have been performed for those and other halogenated phenols (2,4,6-triiodophenol and 2,4,6-trifluorophenol) to clarify which physicochemical parameter can explain better the recognition pattern observed. Finally, the assay has been adapted to the analysis of urine samples. The studies have shown that a limit of detection of 1 microg L(-1) can be accomplished on this biological matrix by combining the ELISA procedure with a C18 solid-phase extraction method.     

Development of highly fluorescent detection reagents for the construction of ultrasensitive immunoassays

We developed two kinds of highly fluorescent streptavidin-based conjugates for use as universal detection reagents in ultrasensitive immunoassays. The direct conjugate was produced by covalently linking streptavidin to poly(Glu: Lys) which was labeled heavily with Eu chelates; the indirect conjugate was made by first conjugating bovine serum albumin (BSA) to poly(Glu:Lys) labeled heavily with Eu chelates and then further linking streptavidin to the conjugate of BSA-poly(Glu:Lys)-Eu chelate. Both direct and indirect conjugates were used to construct a highly sensitive time-resolved fluorometric assay for prostate-specific antigen (PSA). Of two monoclonal antibodies used in the assay, one was coated on the well surface of the microtitration strips, and the other was biotinylated. When 10 microL of sample volume was used, we found that the assay using the indirect conjugate had a detection limit of 0.006 microg/L, which was approximately 5.6-fold more sensitive than the one using Eu chelate directly labeled detection antibody and 6.8-fold more sensitive than the one using Eu chelate-labeled streptavidin. However, the assay that used the direct conjugate was 1.5-fold more sensitive than the one that utilized the indirect conjugate. When 45 microL of sample volume was used, a detection limit of 0.001 microg/L was achieved by using the direct conjugate. This improvement in sensitivity should be equally obtainable for the analytes other than PSA. We further demonstrated that the final immunoassay performance was affected not only by the quality of the streptavidin-based conjugate used but also by the quality of the biotinylated antibody reagent. The universal detection reagents described here are believed to be particularly useful for the construction of ultrasensitive time-resolved fluorometric immunoassays and are potentially applicable in other fields such as immunohistochemistry and nucleic acid detection.     

Development of lateral flow immunoassay system based on superparamagnetic nanobeads as labels for rapid quantitative detection of cardiac troponin I

In this study, superparamagnetic nanobeads (SPMNBs) were used as labels to establish a lateral flow immunoassay system for rapid, quantitative detection of cardiac troponin I (cTnI). First, the immunobeads were prepared by coupling monoclone antibody specific to cTnI onto SPMNBs. Then some factors which may influence the detection sensitivity of this lateral flow immunoassay system were studied, such as the amount of antibody immobilized in the Test line, sample volume and the amount of antibody per SPMNB. Finally, lateral flow test strips for detection of cTnI were established and applied to testing standard samples of different cTnI concentrations, which were also measured by Enzyme-linked Immunosorbent Assay (ELISA) in parallel. Furthermore, testing time and stability of magnetic signal were also investigated in this study. Results showed that this lateral flow test for cTnI had a high detection sensitivity of 0.01 ng/ml with a wide detection range of 5 orders of magnitude. The testing time was less than 15 min, and no significant decline of magnetic signal was observed in 140 days. Therefore, this lateral flow immunoassay system using SPMNB as labels will be a new point of care test (POCT) for rapid, quantitative detection of cardiac troponin I.     

Development and validation of a one-step immunoassay for determination of cadmium in human serum.

A sensitive and simple one-step immunoassay was developed and validated for quantitative determination of Cd(II) in human serum. In this method, a monoclonal antibody that recognizes Cd(II)-EDTA complexes was directly immobilized onto microwell plates. The serum sample containing metallothionein(MT)-bound and non-MT-bound Cd(II) was acidified to displace the Cd(II) from MT. The sample was then treated with metal-free EDTA to convert Cd(II) to Cd(II)-EDTA complexes. A mixture of Cd(II)-EDTA complexes derived from serum samples and Cd(II)-EDTA conjugated with peroxidase enzyme was incubated in the wells to compete for binding sites of the immobilized antibody. After addition of peroxidase substrate, the bound fraction of the enzyme conjugate was measured by a microplate reader, and the signal was inversely proportional to the concentration of the Cd(II) in the sample. The assay limit of detection was 0.24 microg/L, and the effective working range at coefficient of variation of < or = 10% was 0.24-100 microg/L. Analytical recovery of spiked Cd(II), in the concentration range between 0.8 and 50 microg/L, was 97.8 +/- 4.0%. The assay was selective for Cd(II); other metal ions (Mn, Co, Cu, Zn, Mg, Hg, Ca, Ni, Fe, and Pb), tested at concentrations considerably higher than those present in human serum, did not significantly interfere with the assay. The assay results correlated well with those obtained by graphite furnace atomic absorption spectrometry (r = 0.984).     

Development of rapid oral bacteria detection apparatus based on dielectrophoretic impedance measurement method

In this study, a bacteria detection apparatus based on dielectrophoretic impedance measurement (DEPIM) method was demonstrated for rapid evaluation of oral hygiene. The authors integrated a micro electrode chip on which bacteria were captured by dielectrophoresis (DEP), an AC voltage source to induce DEP force, and an impedance measurement circuit to a portable instrument that enables rapid and automated oral bacterial inspection in hospitals and clinics. Special considerations have been made on effects of high electrical conductivity of oral samples on DEP force and DEPIM results. It was shown experimentally and theoretically that using a higher electric field frequency for the DEP bacteria trap and the impedance measurement could realise DEPIM application to bacteria inspection from oral samples with higher conductivity. Based on these investigations, the authors optimised the frequency condition of the DEPIM suitable for inspecting an oral sample along with the design and development of a portable DEPIM apparatus for on-site inspection of oral bacteria. Under the optimised frequency condition, DEPIM results were in good agreement with the conventional culture method showing significant applicability of the DEPIM apparatus for practical rapid oral bacteria inspection.     

Solid-phase immobilized tripod for fluorescent renewable immunoassay. A concept for continuous monitoring of an immunoassay including a regeneration of the solid phase

A new concept of immunoassay based on the use of a trifunctional reagent (tripod) and fluorescence resonance energy transfer (FRET) phenomenon is described. This procedure involves differential steps: (1) the tripod bearing (i) a fluorophore, (ii) a molecule structurally close to the target, and (iii) a linker reacts with the solid phase; (2) the solid phase is further activated with an anti-target antibody labeled with a quencher molecule, generating the decrease of the fluorophore emission via FRET; (3) FRET being distance dependent, the presence of the target by competing with the tripod for binding the quencher-labeled antibody leads to a rise of the fluorescence signal; (4) the solid phase is reactivated simply, by adding the quencher-labeled antibody. This method was evaluated in microtiter plates using the susbtance P as model while fluorescein and TAMRA were used as donor and acceptor, respectively. Results clearly illustrated the interest of the method, by allowing (i) a simple regeneration procedure, without requiring any drastic treatment, (ii) a direct fluorescence measurement onto the solid support, leading to a localized and cumulative signal, (iii) an increase of the signal when detecting the target, unlike classical competitive immunoassays, and (iv) a real-time monitoring of the competition and regeneration steps.     

Separation of 2,6-dimethylnaphthalene from a mixture of its isomers using lithium-incorporated zeolite Y synthesized by rapid crystallization method

Lithium-incorporated zeolite Ys (Li-NaYs) were directly synthesized under a hydrothermal condition from a gel mixture of Li₂O·Na₂O·SiO₂·Al₂O₃·H₂O. It was confirmed by XRD, NMR and elementary analysis, that lithium ions could be introduced onto the ion sites of zeolite Y during the course of crystallization. Usage of lithium citrate as the lithium source is the indispensable factor to achieve the synthesis. In adsorption of mixtures of 2,6-dimethylnaphthalene and its isomers, Li-NaYs exhibited greater adsorptive capacities and higher adsorption selectivities to 2,6-dimethyl naphthalene than NaY. Direct hydrothermal synthesis of Li-incorporated zeolite Y prevents the blockage of pore openings and/or channels during lithium ion-exchange. Furthermore, the difference in adsorptive properties between Li-NaY and Li ion-exchanged NaY is due to Li ions being introduced onto the S₁ ion sites, which can hardly be ion-exchanged, during the hydrothermal synthesis.     

Towards an ultrasensitive method for the determination of metal impurities in carbon nanotubes

Residual catalyst metal nanoparticles remain one of the major obstructions in the utilization of carbon nanotubes (CNTs) in many areas owing to their ability to participate in redox chemistry of biomarkers. Presented here is a comparative study of several techniques for quality control of carbon nanotubes in terms of metallic impurities, namely magnetic susceptibility, electron paramagnetic resonance, energy-dispersive X-ray spectrometry, X-ray photoelectron spectroscopy, and thermogravimetric analysis. It is found that the dc magnetic susceptibility is the most sensitive method such that the difference between two CNT samples that underwent slightly different treatments can be detected, whereas the two samples are indistinguishable by other techniques. Therefore, it is suggested that the most accurate statistical method for quality control of carbon nanotubes is dc magnetic susceptibility, which allows the detection of traces of magnetic metal impurities embedded in purified carbon nanotubes, whereas other methods may provide false "impurity-free" information.     

Device for rapid and agile measurement of diffusivity in micro- and nanochannels

The lack of a viable theory for describing diffusivity when fluids are confined at the micro- and nanoscale [Ladero et al. Chem. Eng. Sci.2007, 62, 666-678; Deen AIChE J.1987, 33, 1409-1425] has necessitated accurate measurement of diffusivity (D) [Jin and Chen Chromatographia2000, 52, 17-21; Nie et al. Science1994, 266, 1018-1021; Durand et al. Anal. Chem.2009, 81, 5407-5412], crucial for a host of micro- and nanofluidic technologies [Grattoni et al. Curr. Pharm. Biotechnol.2010, 11, 343-365]. We demonstrate a rapid and agile method for the direct measurement of diffusivity in a system possessing 10(4) to 10(5) precisely fabricated channels with characteristic sizes (β) ranging from micro- to nanometers. Custom chambers allowed us to measure the diffusivity in a closed unperturbed system using UV/vis spectroscopy. D was measured for rhodamine B (RhoB) in aqueous solution in channels of 200 and 1 μm, as well as 13 and 5.7 nm. The observed logarithmic scaling of diffusivity with β, in close agreement with prior experiments, but far from theoretical prediction, surprisingly highlights that diffusivity is significantly altered even at the microscale. Accurate measurement of D by reducing the size of the source reservoir by 3 orders of magnitude (from 150 μL to 910 nL) proves that a substantial reduction in measurement time (from 7 days to 40 min) can be achieved. Our design thus is ready for rapid translation into a standard analytical tool--useful for multiple applications.     

Development of an optical biosensor based immunoassay to screen infant formula milk samples for adulteration with melamine

The illegal adulteration of milk with melamine in 2008 in China led to adverse kidney and urinary tract effects in hundreds of thousands of children and the reported deaths of six. The milk had been deliberately adulterated to elevate the apparent protein content, and subsequently melamine was detected in many milk-related products which had been exported. This led to the banning of imports of milk and milk products from China intended for the nutritional use of children and to the implementation of analytical methods to test products containing milk products. An optical biosensor inhibition immunoassay has been developed as a rapid and robust method for the analysis of infant formula and infant liquid milk samples. A compound with a chemical structure similar to that of melamine was employed as a hapten to raise a polyclonal antibody and as the immobilized antigen on the surface of a biosensor chip. The sensitivity of the assay, given as an IC(50), was calculated to be 67.9 ng mL(-1) in buffer. The antibody did not cross-react with any of the byproducts of melamine manufacture; however, significant cross-reactivity was observed with the insecticide cyromazine of which melamine is a metabolite. When sample matrix was applied to the assay, a limit of detection of <0.5 μg mL(-1) was determined in both infant formula and infant liquid milk. The development of the immunoassay and validation data for the detection of melamine is presented together with the results obtained following the analysis of melamine-contaminated milk powder.     

Development of an enrichment measurement technique and its application to enrichment verification of gaseous UF6

A technique has been developed to verify the enrichment of gaseous UF₆ inside cylinders. This technique combines an X-ray fluorescent measurement of the total amount of gaseous uranium with a measure of the 185.7 keV gamma ray from ²³⁵U using two collimators to obtain an enrichment that is independent of the pressure of the gaseous uranium and independent of the deposit that forms on a surface in contact with UF₆. This technique has the required sensitivity to determine whether the process gas is of uranium enrichment ≤ 20% or >20%.     

Development of an interferometer for measurement of the diffusion coefficient of miscible liquids

A common-path interferometer (CPI) system was developed to measure the diffusivity of transparent liquid pairs by real-time visualization of the concentration gradient profile. The CPI is an optical technique that can be used to measure changes in the gradient of the refractive index of transparent materials. The CPI is a shearing interferometer that shares the same optical path from a laser light source to the final imaging plane. Molecular diffusivity of liquids can be determined by use of physical relations between changes in the optical path length and the liquid phase properties. The data obtained by this interferometer are compared with similar results from other techniques. This demonstrates that the instrument is reliable for measurement of the diffusivity of miscible liquids and allows the system to be compact and robust. It can also be useful for studies in interface dynamics as well as other applications in a low-gravity environment.     

Development of engineered stationary phases for the separation of carotenoid isomers

A variety of bonded phase parameters (endcapping, phase chemistry, ligand length, and substrate parameters) were studied for their effect on column retention and selectivity toward carotenoids. Decisions were made on how each of these variables should be optimized based on the separation of carotenoid and polycyclic aromatic hydrocarbon test probes. A column was designed with the following properties: high absolute retention, enhanced shape recognition of structured solutes, and moderate silanol activity. These qualities were achieved by triacontyl (C30) polymeric surface modification of a moderate pore size (approximately 20 nm), moderate surface area (approximately 200 m2/g) silica, without subsequent endcapping. The effectiveness of this "carotenoid phase" was demonstrated for the separation of a mixture of structurally similar carotenoid standards, an extract of a food matrix Standard Reference Material, and a beta-carotene dietary supplement under consideration as an agent for cancer intervention/prevention.