Catalytical amplification

Over the past 5 years, a number of researchers have started to explore and mimic these approaches in the laboratory. Enzyme-assisted formation of supramolecular polymers has several unique features. These include selectivity, confinement and catalytic amplification, which allow for superior control as observed in biological systems. These systems are finding applications in areas where supramolecular function is directly dictated by molecular order, for example in designed biomaterials for 3D cell culture, templating, drug delivery, biosensing, and intracellular polymerisations to control cell fate. Overall, biocatalytic production of supramolecular polymers provides a powerful new paradigm in stimuli-responsive nanomaterials. 

Figure 11.21 Catalytic amplification of the DNA detection system. Adapted from Ref. 75 with permission.

The substrate for the indicator reaction, dichloroindophenylbutyrate, is pale yellow. The product of the reaction is deep blue, with an absorbance maximum at 620 nm. The ingenuity of this method lies in the production of an enzyme inhibitor, (oxotrifluor-obutyl)phenol, rather than a species that is directly quantitated. The inhibitor affects the activity of the esterase, resulting in catalytic amplification of the initial alkaline phosphatase activity. The detection limit of the amplified assay has been reported as 3.2 x 10 14M, a 100-fold improvement over the detection limit of the standard alkaline phosphatase assay. 

Enzymes are currently the most widely used and investigated labels for immunoassays, because a single enzyme label can provide multiple copies of detectable species. This catalytic amplification results in immunoassay detection limits that rival those of radioimmunoassay without the storage and disposal problems associated with radioisotopes. Enzyme immunoassays label either ligands or antibodies with enzyme, and enzyme activity in bound or free fractions is measured. Heterogeneous immunoassays employing enzymatic labels have been named enzyme-linked immunosorbent assays (ELISAs). ELISA methods usually employ antibody immobilized onto the wells of polystyrene microtiter plates, and may be... 

Detection limits would be considerably improved (lower) if the indicator reaction generates and inhibitor of a fluorophore-producing enzymatic reaction, because of the additional catalytic amplification provided by this reaction. 

Single event (e.g., 1 photon/reaction) Catalytic amplification Continuous (like substrate)... 

Enzyme immunoelectrodes involve the spatial coupling of the sensor, the immunocomplex, and the catalytic amplification by indicator enzymes. Like the sensor systems described above, enzyme immunoelectrodes are based on common principles of EIA. The choice of enzymes for EIA is rather restricted and is further diminished when electrodes are to be used for detection. So far only GOD, catalase, and HRP have been combined with oxygen-sensing polarographic sensors. An overview of enzyme immunoelectrodes is given in Table 21. 

And enzyme cascade is a powerful mechanism in which a series of enzymes are sequentially activated. Activation is often initiated by a second messenger molecule (signal amplification). The enzyme activated by the second messenger modifies multiple copies of a number of different target enzymes. Those target enzymes that are activated in the process of modification may also modify multiple copies of a second set of target proteins. These expanded enzymatic responses are referred to as catalytic amplification (I = inactive, A = active.)... 

Covalent modification systems can amplify the effect of a regulator both by catalytic amplification and by cascade amplification. 

Catalytic amplification occurs when one enzyme modifies a large number, say TV, of the molecules being controlled. Hence, one molecule of regulator enzyme affects TV molecules of the enzyme being controlled, an amplification factor of TV. For example, MAP kinase activates the protein ribosomal S6 kinase (Rsk) each molecule of MAP kinase can phosphorylate many molecules of Rsk, giving catalytic amplification (Fig. 31.13). 

A cascade multiplies the effects of linked catalytic amplifications to give very rapid massive amplification of small initial events. 

Figure 14-2. Principles of biological transduction and amplification. Binding of an effector at a receptor protein R induces a conformation change which can directly have for consequence the opening of an ion channel (left) with electrolyte influx and membrane depolarization, or initiate the formation of a second messenger (here cyclic adenosine monophosphate, cAMP) by adenylate cyclase, which can then, in catalytic amplification steps, lead to electrochemical or chemical ampliflcation and signal spreading.

Yoshida K, Furuta T, Kawabata T (2010) Perfectly regioselective acylation of a cardiac glycoside, digitoxin, via catalytic amplification of the intrinsic reactivity. Tetrahedron Lett 51 4830- 832... 

Harada Y, Li X, Bohn PW, Nuzzo RG (2001) Catalytic amplification of the soft hthographic patterning of Si. Nonelectrochemical orthogonal fabrication of photoluminescent porous Si pixel arrays. J Am Chem Soc 123 8709-8717... 

Enzymes (and all biological elements, such as tissues, cells, microorganisms, which contain enzymes) represent the class of what are called catalytic elements. Enzyme biosensors have several advantages. These include the catalytic amplification of the biosensor response by modulation of the enzyme activity with respect to the target analyte a stable source of... 

Fig. 3.21 Superimposed C Is and Pt 4f X-ray photoelectron images (top) and the Pt 4f high resolution X-ray photoelectron spectra corresponding to spots a and b bottom). Top image reprinted from Harada, Y., Li, X., Bohn, P. W., Nuzzo, R. G. Catalytic Amplification of the Soft Lithographic Patterning of Si. Nonelectrochemical Orthogonal Fabrication of Photoluminescent Porous Si Pixel Arrays. J. Am. Chem., Soc., 123, 8709 (2001). Copyright 2001 American Chemical Society...

Harada Y, Gitolami GS, Nuzzo RG (2003) Catalytic amplification of patterning via surface-confined ring-opening metathesis polymerization on mixed primer layers formed by contact printing. Langmuir 19 5104-5114... 

Xiao, X. Bard, A. J. 2007. Observing single nanoparticle collisions at an ultramicroelectrode by electro-catalytic amplification. J. Am. Chem. Soc. 129 9610-9612. 

Zhou, H. Fan, F.-R. F. Bard, A. J. 2010. Observation of discrete Au nanoparticle collisions by electro-catalytic amplification using PT ultramicroelectrode surface modification. J. Phys. Chem. Lett. 1(18) 2671-2674. 

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