Rajiv Pande

Biosensors for pesticide detection based on alkaline phosphatase-catalyzed chemiluminescence

Abstract An enzyme-based detection methodology for sensing and quantitation of organophosphorus pesticides is described. The enzyme, alkaline phosphatase, is used in soluble form for reactions in bulk solutions, and in immobilized form for reactions on optical fibers or in glass capillaries. The immobilization strategy involves building a molecular assembly of the enzyme and a conjugated copolymer, poly (3-undecylthiophene-co-3-thiophenecarboxyldehyde-biotin-LC-hydrozone) on a glass surface. Hydrophobic or specific biotin-streptavidin interactions are used to immobilize the biotinylated copolymer on a silanized glass surface and attach a streptavidin conjugate of alkaline phosphatase to the copolymer. Alkaline phosphatase catalyzes the dephosphorylation of a macrocyclic compound, chloro-3-(4-methoxy spiro[1,2 dioxetane-3-2′-tricyclo-“3.3.1.1”-decan]-4-yl) phenyl phosphate and releases light; the chemiluminescence signal is detected with a simple photomultiplier tube. The enzyme activity, and proportionately the chemiluminescence signal strength, are inhibited in the presence of organophosphorus pesticides such as paraoxon. Quantitation of pesticides is thus possible from the inverse correlation between the enzyme activity or signal strength and pesticide concentration. Detection of paraoxon at a concentration of about 50 ppb is achieved with this system. Reaction kinetics are examined for the enzyme in both states. The immobilized enzyme can be reused a number of times, and there is no significant loss of enzyme activity over a period of eight weeks. The design of a flow cell for continuous analysis of the pesticides is described.

Trace Analysis of Zn(II), Be(II), and Bi(III) by Enzyme-Catalyzed Chemiluminescence

A novel technique for the trace analysis of metal ions Zn(II), Be(II), and Bi(III) in bulk solutions is discussed. This technique involves the generation of a chemiluminescence signal from alkaline phosphatase catalyzed hydrolysis of a phosphate derivative of 1,2-dioxetane. Zn(II) can be determined by two methods, reactivation of the alkaline phosphatase apoenzyme and inhibition of the native enzyme. Be(II) and Bi(III) can be determined quantitatively by inhibition of the native enzyme. Subppb to ppm level detection of Zn(II), Be(II), and Bi(III) has been achieved. Initial studies with mixed metals are also reported. The technique described is rapid and sensitive and can be readily applied to the microassay of heavy metal ions.

A chemiluminescence-based biosensor for metal ion detection

Abstract Inhibition of the native metalloenzyme, alkaline phosphatase, in the presence of some metal ions, and the reactivation of its apoenzyme by Zn(II) ions is used to determine metal ion concentrations. Alkaline phosphatase-catalysed hydrolysis of a chemiluminescent substrate, chloro 3-(4-methoxy spiro [1,2-dioxetane-3-2′-tricyclo-[3.3.1.1]-decan]-4-yl) phenyl phosphate, generates light. By measuring the chemiluminescence signal strength in the presence or absence of metal ions, this reaction can be used to detect and determine metal ion concentrations. The immobilization of alkaline phosphatase on different glass surfaces by covalent coupling using a bifunctional reagent, glutaraldehyde, was demonstrated. Using chemiluminescence measurements, Zn(II), Be(II) and Bi(III) were detected in trace levels. This technique forms the basis in the development of a metal ion-based fibre optic sensor.

DNA bound to polypyrrole films : high-resolution imaging, DNA binding kinetics and internal migration

We have studied the time-dependent uptake of 35S radiolabeled DNA with electrochemically prepared polypyrrole films. The two distinct polypyrrole film surfaces, a rough (solution polymeric growth face, R) and a smooth surface (electrode face, S) were characterized by low-resolution AFM and high-resolution transmission electron microscopy (TEM). These studies showed the presence of steep contours and defects in the form of large and small surface holes and valleys on the rough surface of polypyrrole. The void dimensions ranged from the nanoscale to micron size. By contrast, the smooth surface was flatter and largely devoid of significant structural defects and exhibited closer packing of the polypyrrole chains over large areas. Both surfaces were comprised largely of chains whose average diameters were 1.0-1.2 +/- 0.3 nm. The surface characterization studies were complemented by time-dependent DNA uptake studies which showed a t1/2-dependent total uptake of 35S DNA at higher levels on the rough surface compared to the smooth surface. This is consistent with the apparent higher effective surface area of the rough surface compared to the smooth. Using a proportional counter the time-dependent ratio (R/S) of the 35S DNA detected from the rough surface of the polypyrrole disk to that detected from the smooth surface suggested that DNA was migrating into the disk interior from its uptake surface. The rough side defect dimensions measured by TEM were more than sufficient to allow for the penetration and migration of DNA into the disk interior. Both R/S ratios were extrapolated and found to intersect at an R/S value close to 1.0, suggesting a kinetic process leading ultimately towards a nearly uniform radiolabeled DNA distribution in the disk. These kinetic results were in agreement with the surface characterization studies and suggest a model in which sizeable internal pores exist throughout the electrochemically prepared polypyrrole, that could account for the DNA migration effect. This was confirmed by TEM of the interior of a polypyrrole disk produced by Argon ion milling.

Intelligent Materials Properties of DNA and Strategies for Its Incorporation into Electroactive Polymeric Thin Film Systems

We propose to create a novel class of intelligent materials by integrating two separate classes of intelligent materials—one biological and the other a thin film conducting polymer. The first class, DNA possesses superior intelligent material properties designed over evolutionary time to function specifically and efficiently in integrated macromolecular arrays in cells called chromo somes. The second material is the polymeric thin film or two-dimensional Langmuir Blodgett (LB) monolayer film. In our approach, films will be comprised of electroactive alkylated conducting polymeric materials, such as polyalkylpyrrole and polyalkylthiophene, that are derivatized with biotin. Steptavidin conjugated DNA will be attached directly or biotinylated DNA will be stably at tached to this film via a bridging streptavidin protein. To date, the bulk of our work has centered on characterizing the DNA binding to thick films of conducting polymers. A near term aim is to incor porate this signal transduction system into fiber optic biosensors for specifically detecting nucleic acid analyte. Our ultimate aim is to create novel ordered structures possessing unique integrated in telligent functions which respond to their environment and provide signal transduction approaches via their electronic and optical functions.

Comparison of Single and Double Stranded DNA Binding to Polypyrrole

Abstract : The polycation conducting polymer, oxidized polypyrrole, possesses the ability to form complexes with DNA. Our previously proposed diffusion limited binding model for double helical DNA was also found to be applicable to single stranded DNA in this study. However, single stranded DNA was found to bind PPy at a higher level than double helical DNA. An investigation of electropolymerized PPy film morphology using SEM and TEM techniques revealed two distinctly differing surface morphologies for the Platinum (Pt) electrode face (smooth) and polymeric growth face (rough). The DNA uptake levels were found to be consistently different on either surface. Double stranded DNA penetration into the surface increased with extended time periods of exposure while a similar phenomenon, but to a lower extent, was observed for single stranded DNA. DNA, Polypyrrole, Conducting polymers.

Integrating biotinylated polyalkylthiophene thin films with biological macromolecules: biosensing organophosphorus pesticides and metal ions with surface immobilized alkaline phosphatase utilizing chemiluminescence measurements

We describe a methodology for immobilizing the enzyme alkaline phosphatase onto a glass surface using a novel biotinylated copolymer poly (3-undecylthiophene-co-3- thiophenecarboxaldehyde) 6-biotinamido hexanohydrazide attached hydrophobically to silanized glass. The biotin-streptavidin protein interaction is used to carry out this immobilization. Alkaline phosphatase catalyzes the dephosphorylation of a class of macrocyclic compounds: including CSPD {chloro 3-[4-methoxy spiro(1,2 dioxetane-3-2-trichloro-(3.3.1.1)-decan]-4 yl}phenyl phosphate to a product species which emits energy by chemiluminescence. We can detect this chemiluminescence signal with a photomultiplier tube for both enzymatic catalysis in solution and the surface immobilized enzyme (streptavidin conjugate). This enzyme is inhibited by the organophosphorus class of pesticides as well as nerve agents. The enzyme is also inhibited by Be(II), Bi(III) as well as excess Zn(II), while the apoenzyme is reactivated by Zn(II). We demonstrate in this study that two representative organophosphorus pesticides inhibit the enzymatic production of chemiluminescent products. This is true for the enzyme conjugate both free in solution and immobilized. We can detect pesticides down to about 50 ppb for the enzyme in solution and 500 ppb for surface immobilized enzyme in a 100 (mu) l capillary. Detection of Zn(II) by apoenzyme reactivation occurs down to 3 ppb. Be(II) and Bi(III) are detected by inhibition down to 1 ppm.

Biotinylated polyalkylthiophene thin films and monolayers that specifically incorporate phycobiliproteins: toward smart materials

We are investigating thin film and monolayer systems that involve conjugated conducting polymers and specific biological macromolecules. One class of conducting polymers, polyalkylthiophenes, are derivatized with biotin. These biotinylated polymers form the basis for a generic cassette system of attachment for any biological molecule through biotinylation or interaction with streptavidin. The high affinity of the biotin-streptavidin system, used in sequential steps, forms the basis of the cassette method. We have formed both monolayers and thin films (a few nanometers) of the cassette assembly in which phycobiliproteins are incorporated. We are investigating the optical signal transduction properties of specific phycobiliproteins (phycoerythrin, phycocyanin and allophycocyanain) using the cassette system on the inner surface of glass capillaries and on optical fiber surfaces. Phycobiliprotein photocurrent signals in conducting polymer matrices on microelectrodes are also being investigated. Our aim is to integrate the signal transduction mechanisms of the phycobiliproteins within monolayers or thin films of the conducting polymers to create biosensors and related smart materials for applications in biomedicine and biotechnology.

Interfacing Conducting Polymers and Biological Macromolecules: A Case Study of Insecticide Biosensor Development

The creation of smart materials is a current goal of many laboratories including our own. We are involved in studying a number of thin film and monolayer systems which share the common features of involving highly conjugated conducting organic polymers and the evolved properties of specific biological macromolecules. In this report we describe a generic ‘cassette’ methodology for immobilizing biotinylated biological macromolecules to hydrophobic surfaces using a novel class of conducting copolymers of polythiophene. These copolymers are derivatized with long alkyl chains and biotin moieties to bind, respectively, to the hydrophobic surface and the biotinylated species, through the biotin - streptavidin interaction. We utilize the monolayer ‘cassette’ approach to attach a signal transducing biomolecule alkaline phosphatase to the surface of a glass capillary. This produces a flexible system in which chemiluminescence provides the basis for a useful measurement strategy. A novel technique involving the generation of chemiluminescence signal from alkaline phosphatase catalyzed dephosphorylation of a macrocyclic phosphate compound is described. Detection of paraoxon and methyl parathion, both enzyme inhibitors, has been achieved at ppb levels. The technique is rapid, sensitive, and is applicable to the detection of all organophosphorus based insecticides. The technique will be used in developing a fiber;optic biosensor for remote detection of insecticides.