Protein detection schemes

Figure 12.13 A DNA/Au nanopaiticle-based protein detection scheme, (a) Preparation of hapten-modified nanopaiticle probes, (b) Protein detection using protein binding probes. Notice that there are nine GC pairs in sequence A, and there are only two GC pairs in sequence B. (Reproduced with permission from J.-M. Nam et al., J. Am. Chem. Soc. 2002, 124, 3820-3821. Copyright 2002 American Chemical Society.)...

In addition to the solution-phase protein detection assays such as biobarcode assays, a surface-based protein detection method was developed by Niemeyer and Ceyhan (70). In this work, a biotin-labeled antibody was conjugated to DNA using streptavidin as a tinker molecule. This conjugate was hybridized to complementary DNA-AuNPs and antibody-functionalized AuNPs. These particles are allowed to form a sandwich structure with a target protein and a second antibody immobilized on a flat surface. This was followed by a silver enhancement step catalyzed by the AuNPs which resulted in a LOD of 50 fmol (200 pM). This method is conceptually similar to the corresponding surface-based scanometric detection of DNA. Further work demonstrated a similar protein detection scheme without the silver enhancement (71). Instead, multiple layers of secondary DNA-AuNPs were used to increase signal enhancement [LOD = 0.1 finol (2 pM)]. 

Without highly specific binding to proteins on the chip, thorough washing of the array may remove specific binders as well as non-specific binders. Protein bindings cannot offer a universal detection scheme like hybridization used in DNA arrays, which makes it difficult to discover enough probes for proteomics. 

PPEs in water and prevent aggregation and self-quenching [53]. Heeger and co-workers paired anionic PPV with a nonconjugated cationic polymer to form a charge-neutral complex and saw both reduction of nonspecific effects and some loss in sensitivity in protein detection [54]. Polyelectrolytes may be best used in assay schemes, such as DNA hybridization assays or drug discovery screens, where the assay conditions are well controlled and nonspecific interactions reduced or avoided. 

As illustrated in Figure 2B, these interface detection schemes provide a means of obtaining simplified spectra containing only resonances of interface residues within a large membrane protein complex, to obtain... 

Figure 8.14 Scheme depicting the analytical procedure for biodetection based on catalytic H202 reduction at PtNPs (a) Detection probe modified PtNPs for DNA detection (b) thrombin aptamer DNA sequence modified PtNPs for thrombin protein detection.69 (Reprinted with permission from R. Polsky et al., Anal. Chem. 2006, 78, 2268-2271. Copyright 2006 American Chemical Society.)... 

Figure 5.21 Representation of immunoassay-based detection schemes. A reagent protein immobilized on the sensor substrate binds the compiementaiy target protein (Step 1 - direct detection). In amplified mass immunosorbent assay (AMISA), a conjugate enzyme is added to form a bound sandwich complex (Step 2). that can react with other reagents to amplify the mass increase on the sensor surface. (Adapted with peraiission. See Ref. [263], 1988 American Chemical Society.)...

From an experimental perspective the major challenge for future quantum interferometry with proteins or nanocrystals is the development of a suitable source, which provides a sufficiently intense beam of slow, massive, well-collimated, internally cold, neutral particles. Also novel detection schemes for such objects are currently being investigated, since the current technique of thermal ionization cannot be applied to thermolabile molecules. 

As is visible from Fig. 11, proton spins are not only more abundant than or nuclei in proteins but ( H- H) contacts provide key constraints defining secondary and tertiary protein structure. Because of the hmited spectral resolution of MAS NMR spectroscopy, indirect detection schemes have been employed for a long time... 

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