An immunochromatographic dipstick as an alternate for monitoring of heroin metabolites in urine samples.

Heroin use and addiction pose serious risks and side effects due to overdose. Quantification of heroin in biological samples is challenging due to rapid deacetylation of heroin to its active metabolites. In this study, we report the quantification of metabolic degradation of heroin by-products in biological urine samples. The presence of the drug was monitored after oral administration of heroin at different time intervals. Various biophysical techniques, such as high performance liquid chromatography (HPLC) and mass spectrometry (MS) were used to evaluate the presence of the drug. A competitive fluorescence based immunoassay was developed with a limit of detection (LOD) up to 0.01 ng mL-1 and the IC50 value was 0.1 ng mL-1, while the dipstick assay shows a LOD up to 5 ng mL-1. Rapid detection of narcotic drugs was carried out for biological urine samples collected at various time points. Validation of the developed dipstick was carried out for the standard as well as the spiked urine samples by fluorescence based immunoassay (FIA), using anti-morphine antibodies. A strong correlation (R = 0.94) was obtained between the developed dipstick and FIA assay for biological urine samples collected at various time points. The developed immunochromatographic dipstick is highly sensitive, field applicable and cost effective, and can serve as a first choice for the monitoring of narcotic drugs in blood, urine and saliva in drug addicts and athletes.

One step in-situ synthesis of amine functionalized graphene for immunosensing of cardiac marker cTnI.

2-Aminobenzyl amine (2-ABA) functionalized graphene is proposed for the ultrasensitive immunosensing of Cardiac Troponin I (cTnI). 2-ABA was electrochemically polymerized on the graphene decorated interdigitated electrode to obtain the amine functionalized graphene (f-GN). The f-GN electrode was then modified with monoclonal anti-cTnI antibodies via Schiff reaction based chemistry. Detailed characteristics of the processes involved and the finally developed antibody conjugated f-GN interdigitated electrode have been studied. The above micro-device was used in a drain source configuration for the sensing of cTnI. A wide dynamic linear range of antigen detection (0.01-1ng/mL) is achieved with the limit of detection of 0.01ng/mL. The utility of the proposed sensing technique is demonstrated by successfully testing the antigen concentration in spiked serum samples.

Isolation and characterization of an atrazine-degrading Rhodococcus sp. strain MB-P1 from contaminated soil.

AIMS: The aim of this study is to isolate and characterize organisms capable of utilizing high concentration atrazine from the contaminated sites. METHODS AND RESULTS: A selective enrichment was used for isolating atrazine-degrading organisms from the contaminated sites resulting in isolation of an efficient atrazine-degrading organism designated as strain MB-P1. On the basis of 16S rRNA gene sequencing, total cellular fatty acid analysis and physiological and biochemical tests, strain MB-P1 was identified as a member of genus Rhodococcus. High performance liquid chromatography was performed to identify the atrazine degradation intermediates demonstrating that the degradation proceeds via formation of 'de-ethylatrazine' and 'de-isopropylatrazine'. Further, plasmid curing by SDS method showed atrazine-degrading gene(s) to be plasmid-encoded. CONCLUSIONS: We have successfully isolated a Rhodococcus sp. strain MB-P1 which is capable of utilizing atrazine as sole source of carbon and energy at very high concentrations of 1000 ppm. The pathway for degradation of atrazine has also been determined. The metabolic gene(s) responsible for atrazine degradation was found to be plasmid-encoded. SIGNIFICANCE AND IMPACT OF THE STUDY: Rhodococcus sp. strain MB-P1 could be used as an ideal model system for in-situ degradation and restoration of ecological niches which are heavily contaminated with atrazine.

Isolation of a member of Acinetobacter species involved in atrazine degradation.

Contribution of Acinetobacter genus in the degradation of atrazine and its analogs is reported here. An interesting bacterial isolate capable of degrading atrazine as high as 250 ppm was isolated from a soil heavily contaminated with atrazine. The permissible level of atrazine in drinking water is 3 ppb and hence use of a strain capable of atrazine degradation as high as 250 ppm would be of immense help for rapid environmental cleanup. This isolate was found to be capable of best growth at 37 degrees C and at pH inclined towards the alkaline side. It was found that atrazine was utilized as a carbon and not as a nitrogen source. Acinetobacter species was also active on other triazine pesticides, viz., simazine, terbutryn, cyanazine, and prometon. There are very few reports on the degradation of atrazine by any member of this genus and hence this could lead to new degradation pathways and new metabolites.

Activating piezoelectric crystal surface by silanization for microgravimetric immunobiosensor application.

The development of a microgravimetric immunobiosensor using a piezoelectric quartz crystal as a detector requires a stable and reproducible immobilization method for ligand binding. The method of silanization using 3-aminopropyltriethoxysilane (APTES) has been widely used for activating the carrier surface. In the present study, APTES deposition on a piezoelectric crystal surface was studied under various solvent conditions. A fluorescence method, using fluorescence isothiocyanate as a dye, was demonstrated for the quantification of amino groups on the silanized piezoelectric crystal surface. The optimum binding conditions of APTES deposition on a piezoelectric crystal surface were incorporated for the covalent immobilization of protein on the crystal surface in developing a stable and sensitive microgravimetric immunobiosensor. Determination of immunoglobulin G (IgG) concentration was performed using APTES modified piezoelectric crystals coated with protein G. The resonant frequency shift, resulting from the formation of protein G-IgG complex on the crystal surface, correlated with the concentration of IgG in the range 10 ng/ml to 0.1 mg/ml. The APTES modified, protein G coated crystal were found to be quite stable and did not show a significant loss of sensitivity even after 12 weeks of storage at 4 degrees C in a desiccator.

Development of piezoelectric crystal based microgravimetric immunoassay for determination of insulin concentration.

A microgravimetric, piezoelectric crystal based immunoassay for the quantification of insulin concentration is described. The method utilizes a modified piezoelectric crystal device having an antibody specific to insulin bound to its surface. The antibody to insulin was immobilized on the surface of crystal electrode by using either 3-aminopropyltriethoxy silane (3-APTES), polyethyleneimine (PEI) or covalently coupled protein A-gold immobilization method. Coating an electrode with a cross linked protein A-antibody complex gave better results in terms of sensitivity and stability. Using the system described, the insulin concentration up to 1 ng ml-1 could be detected. The stability and reusability of the system was further improved by using a mild eluting reagent which successfully removed the bound insulin molecules from the antibody-coated crystal without affecting the immobilized insulin antibody. Scanning tunneling microscopic (STM) study was also done to confirm the surface coverage and orientation of insulin and antibody molecules on the modified piezoelectric crystal electrode surface. A comparison between the present study and the well-established radioimmunoassay technique (RIA) revealed that the described microgravimetric immunoassay technique (MIA) could successfully be developed as an alternative of RIA.

Determination of immunoglobulin M concentration by piezoelectric crystal immunobiosensor coated with protamine.

In the present study, the specific binding between protamine and immunoglobulin M (IgM) has been exploited to construct a piezoelectric crystal based immunobiosensor for the determination of concentration of IgM. The system consisted of highly stable IC based oscillator, 8-digit frequency counter and modified piezoelectric crystal device. The crystal surface was physically modified and chemically treated (refluxed) with strong acid to produce stable hydroxylic groups of silicon oxide. This modified surface reacted strongly with coupling reagents for binding of protein molecules. The protamine was immobilized by using either gamma-aminopropyltriethoxy silane (gamma-APTES) or 2.2.2-trifluoroethanesulfonyl chloride (tresyl chloride). Scanning electron microscope images of piezo crystal revealed that tresyl activated surface presented more surface area for binding than gamma-APTES modified surface and showed better sensitivity. This immobilization technique also improved the reproducibility and long term stability of the detection system. Using the system described, the IgM concentration up to the level of 10 ng/ml could be detected without interference of IgG.