Limitations in the use of horseradish peroxidase as an enyzme probe in the development of a homogeneous immunoassay for aflatoxin B1

Horseradish peroxidase (HRPO) was used as a probe to quantitate aflatoxin B₁ by a homogeneous immunoassay. The conjugation of AFB₁ to HRPO resulted in 54% loss of enyzme activity. In the presence of AFB₁ specific antibodies, the HRPO-AFB₁ conjugate showed reversal of its lost enzyme activity by 12%. This positive modulatory effect of antibody on the enzyme activity was used as an analytical tool to quantitate AFB₁. The homogeneous assay carried out with free AFB₁ and HRPO-AFB₁ conjugate in the presence of antibodies indicated poor linearity as compared to the heterogeneous assay. It was observed that the number of HRPO-lysine residues involved in AFB₁ conjugation were 6–8. The low level of modulation of enzyme activity by antibody with respect to HRPO-AFB₁ conjugate, could possibly be attributed to the limited number of lysine residues in the HRPO molecule and its proximity to the active site of the enzyme. Thus, HRPO was found to be limiting as an enzyme with respect to the homogeneous enzyme immunoassay for AFB₁ analysis. The antibodies raised were specific for AFB₁, and showed excellent linearity even at high dilution for the detection of AFB₁ by ELISA indicating that antibodies per se were not the limiting factor.     

Microscale heat transfer in an evaporating moving extended meniscus

The evaporative heat flux distribution in the leading edge region of a moving evaporating thin liquid film of pentane on quartz was obtained by analyzing the measured thickness profile for thicknesses, δ < 2 μm. The profiles in a constrained vapor bubble were obtained using image analyzing interferometry. Although the evaporating meniscus appeared to be benign (i.e., without additional observed motion beyond creeping), high heat fluxes were obtained. Significantly higher heat fluxes are possible. The interfacial slope, curvature, interfacial shear stress, and liquid pressure profiles were also obtained. Results obtained using a continuum model were consistent with those obtained using a control volume model. The measured pressure field profile of the isothermal extended meniscus agreed with the constant pressure field predicted by the augmented Young–Laplace model. For the non-isothermal case, measured thickness gradients lead to disjoining pressure and curvature gradients for fluid flow and evaporation. The experimental results demonstrate that disjoining pressure at the contact line controls fluid flow within an evaporating completely wetting thin curved film and is, therefore, a useful boundary condition. However, in small interfacial systems, non-idealities can have a dramatic effect.     

Quantitative evaluation of 28 mineral elements by inductively coupled plasma/mass spectrometry and its application in source identification of Indian opium

An analytical method based on inductively coupled plasma/mass spectrometry (ICP/MS) was developed for the determination of 28 mineral elements (Cr, Mn, Co, Ni, Cu, Zn, Ga, Sr, Cd, Ag, Ba, Pb, Bi Y, La, Ce, Nd, Pr, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu) in Indian opium samples. The detection limits were found to be in the range of 0.0008-0.45 ng/mL. The recoveries of spiked samples for each element were found to be in the range of 83-106%, with a precision of less than 9%. A total of 124 opium samples from India were analyzed for the distribution pattern of the 28 mineral elements. Quantitative elemental data were subjected to chemometric analysis in order to determine an optimal classifier to evaluate the source of Indian opium. The study indicated that mineral elements might not be the suitable discriminators for the discrimination of licit opium-growing divisions of India. However, the methodology developed and the analytical data on elemental profile may find important forensic application in discriminating Indian opium with that of licit and illicit opium originating from different geographical regions of world.     

Application of capillary zone electrophoresis in the separation and determination of the principal gum opium alkaloids

We describe the use of capillary zone electrophoresis (CZE) for the qualitative and quantitative determination of major alkaloids (i.e., thebaine, codeine, morphine, papavarine and narcotine) in gum opium involving the analysis of alkaloids without derivatization or purification. Three extractions with 2.5% w/v aqueous acetic acid quantitatively extracted major alkaloids. The separation was carried out by CZE using a 7:3 mixture of methanol and sodium acetate (100 mM, pH 3.1) at a potential of 15 kV, with UV detection at 224 nm. Spiking of pure reference alkaloid standards in the opium extract was used for peak identification. The influences of buffer composition, pH and voltage on the separation of alkaloids were studied. The detection limit of each alkaloid dissolved in methanol was found to be 850 ng/mL (morphine), 450 ng/mL (thebaine), 500 ng/mL (codeine), 550 ng/mL (papaverine), and 500 ng/mL (narcotine) at an injection pressure of 300 mbar (injection volume, 4 nL) with a signal-to-noise ratio of 3:1. The external standard method was used for the quantification of alkaloids. The calibration plot was based on linear regression analysis. The relative standard deviation (RSD) for peak area and migration time was in the range of 1.03-3.56% and 0.34-0.69%, respectively. Percentage compositions (g%) of opium alkaloids in five gum opium samples were found to be in the range of 14.45-15.95 (morphine), 2.0-3.45 (codeine), 1.32-2.73 (thebaine), 0.92-2.37 (papavarine), and 3.85-5.77 (narcotine). The method developed is suitable for the routine analysis of major gum opium alkaloids in samples of forensic importance.     

Limitations in the use of horseradish peroxidase as an enyzme probe in the development of a homogeneous immunoassay for aflatoxin B1

Horseradish peroxidase (HRPO) was used as a probe to quantitate aflatoxin B₁ by a homogeneous immunoassay. The conjugation of AFB₁ to HRPO resulted in 54% loss of enyzme activity. In the presence of AFB₁ specific antibodies, the HRPO-AFB₁ conjugate showed reversal of its lost enzyme activity by 12%. This positive modulatory effect of antibody on the enzyme activity was used as an analytical tool to quantitate AFB₁. The homogeneous assay carried out with free AFB₁ and HRPO-AFB₁ conjugate in the presence of antibodies indicated poor linearity as compared to the heterogeneous assay. It was observed that the number of HRPO-lysine residues involved in AFB₁ conjugation were 6–8. The low level of modulation of enzyme activity by antibody with respect to HRPO-AFB₁ conjugate, could possibly be attributed to the limited number of lysine residues in the HRPO molecule and its proximity to the active site of the enzyme. Thus, HRPO was found to be limiting as an enzyme with respect to the homogeneous enzyme immunoassay for AFB₁ analysis. The antibodies raised were specific for AFB₁, and showed excellent linearity even at high dilution for the detection of AFB₁ by ELISA indicating that antibodies per se were not the limiting factor.