An Ultra-Sensitive Electrochemical Enzyme Immunoassay for Thyroid Stimulating Hormone in Human Serum

A sensitive heterogeneous electrochemical enzyme immunoassay has been developed for thyroid stimulating hormone (TSH) by modifying a commercially available two-site immunoenzymometric assay. p-Aminophenyl phosphate (PAPP) was used as the substrate of alkaline phosphatase, and hydrolysed to p-aminophenol (PAP). The amount of PAP produced from the assay was proportional to the amount of TSH in the sample. Detection of PAP was done by oxidative amperometry in a flow injection system. The working electrode was a glassy carbon electrode whose potential was held at +325 mV (vs Ag/AgCl). The amperometric detection of PAP required only 1 μl of sample (the range of linearity: 50.0 fmol-100 pmol PAP, the limit of detection: 10.9 fmol PAP). Intra-assay precision over the assay range of linearity (0.02–60 mIU l−1 or 0.02–60 pIU TSH) showed a maximum RSD of 8.0%, and a low detection limit of 0.01 mIU l−1 or 0.01 pIU TSH. The study also indicates that this two-site electrochemical enzyme immunoassay correlates well with the Bio-Rad\u27s immunoradiometric assay currently used in our medical center (r = 0.992, slope = 1.53, n = 43) and a highly sensitive immunochemiluminometric assay in the Nichols Institute (r = 0.986, slope = 0.499, n = 23)

Indirect Ultraviolet Detection of Biologically Relevant Organic Acids by Capillary Electrophoresis

Indirect UV detection of fourteen short-chain organic acids (e.g., oxalic acid, citric acid, malonic acid, tartaric acid, methylmalonic acid, α-ketoglutaric acid, succinic acid, ethylmalonic acid, methylsuccinic acid, glutaric acid, apidic acid, methylglutaric acid, lactic acid and pyruvic acid) by capillary electrophoresis is described. The method used phthalate as the UV-absorbing additive in carbonate buffer and the non-absorbing analytes were detected indirectly at 230 nm. The influences of buffer pH, ionic strenght, concentration of phthalate and organic modifier on indirect signal response and migration behavior of the organic acids were investigated. Comparisons of reproducibility on migration time, limit of detection and separation efficiency among three types of capillary (e.g., polyacrylamide-coated, myristyltrimethylammonium bromide-coated and uncoated capillaries) were conducted. The method developed was applied to detect succinic acid, methylmalonic acid, citric acid, glutaric acid and lactic acid in human body fluids, and preliminary results were provided

Capillary Electrophoretic Enzyme Immunoassay for Digoxin in Human Serum

The combined use of capillary electrophoretic (CE) separation and homogeneous enzyme immunoassay for analyzing drugs in hemolyzed, lipemic, or icteric serum samples was investigated. An FDA-approved EMlT assay kit for digoxin in human serum was used. After the enzyme immunoassay, the enzymatic reaction product (NADH) and remaining substrate (NAD+), together with internal standard (p-nitrophenol, NP), were electrokinetically injected into a polyacrylamide-coated electrophoresis capillary and separated under applied potential. Detection was made by monitoring the UV adsorption at wavelengffi of 260 nm. The digoxin level in human serum was determined by comparing the peak area ratio of NADH and NP to the ratios established by the known digoxin standards. In this study, the factors that influence the CE separation were also investigated. Under the optimum conditions, NADH, NAD+, and NP were separated at electric field strength of 438 V/cm in the coated capillary (100 pm x 57 cm) with 200 mM Tris-borate bder (pH 7.9) containing 0.2% hydroxypropyl methylcellulose. CE analyses of serum samples spiked with NADH standards at concentrations of 100 and 400 pM resulted in detection variabilities of less than 2% and analytical recoveries of 98-102%. Both an internal calibration plot for NADH and a dose-response curve for digoxin in serum were constructed. Calibrator serum, patients’ sera with hemolyzed, lipemic, and icteric interference factors, and other pigmented blood components (e.g., serum albumin, bilirubin, hemoglobin, uric acid, coproporphyrin, melanin, protoporphyrin IX, and uroprophyrin) demonstrated no interference in this method. The authors believed that this method is useful for analyzing digoxin in hemolyzed, lipemic and icteric blood samples that are known to create problems in conventional EMIT assays and may be applicable to other EMIT- based assays for monitoring drugs in complex biological matrices

Michaelis-Menten Analysis of Alkaline Phosphatase by Capillary Electrophoresis Using Plug-Plug Reaction

This work evaluated the use of an alkaline phosphatase plug, slowly migrating in a polyacrylamide-coated capillary filled with buffer/soluble polymer, to convert p-nitrophenylphosphate (which was injected into the capillary as a separate plug, and migrated faster than the enzyme) to p-nitrophenol under a constant applied potential. The elution of assay components was monitored on-capillary at 230 nm. The initial reaction velocity of the enzymecatalyzed reaction was estimated from the peak area ratio of the enzyme product to the internal standard. Using the Lineweaver-Burk plots, an average Michaelis constant (KM) for alkaline phosphatase was calculated to be 4.8 ± 0.3 mM (n = 4, CV = 6%). With the constant potential electrophoresis (8 kV/57 cm), the method had a detection limit of 4.4 × 10−5 IU for alkaline phosphatase and a linear range up to 2.1 × 10−3 IU

Indirect Ultraviolet Detection of Biologically Relevant Organic Acids by Capillary Electrophoresis

Indirect UV detection of fourteen short-chain organic acids (e.g., oxalic acid, citric acid, malonic acid, tartaric acid, methylmalonic acid, α-ketoglutaric acid, succinic acid, ethylmalonic acid, methylsuccinic acid, glutaric acid, apidic acid, methylglutaric acid, lactic acid and pyruvic acid) by capillary electrophoresis is described. The method used phthalate as the UV-absorbing additive in carbonate buffer and the non-absorbing analytes were detected indirectly at 230 nm. The influences of buffer pH, ionic strenght, concentration of phthalate and organic modifier on indirect signal response and migration behavior of the organic acids were investigated. Comparisons of reproducibility on migration time, limit of detection and separation efficiency among three types of capillary (e.g., polyacrylamide-coated, myristyltrimethylammonium bromide-coated and uncoated capillaries) were conducted. The method developed was applied to detect succinic acid, methylmalonic acid, citric acid, glutaric acid and lactic acid in human body fluids, and preliminary results were provided

Multi-messenger Observations of a Binary Neutron Star Merger

International audienceOn 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of $\sim 1.7\,{\rm{s}}$ with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg(2) at a luminosity distance of ${40}_{-8}^{+8}$ Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 $\,{M}_{\odot }$. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at $\sim 40\,{\rm{Mpc}}$) less than 11 hours after the merger by the One-Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ∼10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position $\sim 9$ and $\sim 16$ days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC 4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta