Signal Amplification and Detection of Small Molecules via the Activation of Streptavidin and Biotin Recognition

Abstract

Molecular recognition (e.g., antigen–antibody, DNA–DNA, and streptavidin–biotin) is a generic, yet highly versatile and powerful strategy employed in enzyme-catalyzed signal amplification process. However, this approach is not applicable to metals, anions, and small reactive species (e.g., O2 – and F–), as these molecules are too small to bind effectively to the macromolecules. In this paper, we demonstrate an enzyme-catalyzed signal amplification approach based on the controlled binding between streptavidin and target activated affinity-switchable biotin (ASB) probes, for the detection of O2 – and F–, using electrochemical and fluorescent detection techniques. The underlying rationale behind this design is that, while the ASB probe would not bind with the streptavidin–enzyme conjugate due to its low binding affinity with streptavidin, in the presence of the target analyte, the ASB probe on the immobilized surface will be activated to form biotin, which can then bind with the enzyme-tagged streptavidin to initiate signal amplification process. This versatile approach can also be applied in the imaging of endogenously secreted O2 – along the plasma membrane of living cells using streptavidin conjugated with multiple fluorescent dye reporters. We believe that this ASB probe strategy will be useful for a wide range of applications, such as in basic biological research and medical diagnoses, where highly specific signal enhancement is required.

Mechanistic Investigation on Copper–Arylacetylide Polymerization and Sensing Applications

“…For example, the biotin can form strong non‐covalent interactions with streptavidin with a dissociation constant of 10 −15 mol L −1 . Streptavidin has been widely used to tag proteins for signal amplification in immunoassay [18] . Therefore, the P 3 can be used to recognize streptavidin‐conjugated biomolecules through the streptavidin‐biotin binding.…”

Mechanistic Investigation on Copper–Arylacetylide Polymerization and Sensing Applications

“…[17] Thus the Cu-arylacetylide polymerization reaction offers potentials for the preparation of ultra-thin membranes and functionalization of substrate surfaces.T o explore the tolerance of the reaction, arylacetylide with biotin groups (M2), PEO (M3), and -COOH were synthesized and used to copolymerize with M1.Aseries of poly(Cu-arylacetylide)s with biotin groups (P3), PEO (P4), and -COOH (P5) have been successfully synthesized with M n of 5.96 10 4 gmol À1 for P3,6 .87 10 4 gmol À1 for P4,a nd 4.97 10 4 gmol À1 for P5 at À20 8 8C, which indicates that the Cuarylacetylide polymerization reaction can tolerate various polar groups.T herefore,the Cu-arylacetylide polymerization reaction can be used to prepare various materials with desirable properties.F or example,the biotin can form strong non-covalent interactions with streptavidin with adissociation constant of 10 À15 mol L À1 .S treptavidin has been widely used to tag proteins for signal amplification in immunoassay. [18] Therefore,t he P3 can be used to recognize streptavidinconjugated biomolecules through the streptavidin-biotin binding.I tc an be concluded that the Cu-arylacetylide polymerization distinguishes other polymerization methods with the following distinctive characteristics:(i) mild reaction temperature (À20 8 8Ct o3 08 8C), (ii)a ir atmosphere reaction without protecting gas,( iii)h igh molecular weight (M n > 1000 000), (iv) low PDI (PDI < 1.1), (v) fast polymerization rate,( vi)i mprecise molar ratio between monomers,( vi) tolerance of various polar and functional groups.…”

Ultrasensitive Detection of Ribonucleic Acid Biomarkers Using Portable Sensing Platforms Based on Organic Electrochemical Transistors

“…Biotin on the nanoprobes can specially link to the streptavidin (SA) on the tail of the probe DNA with the binding rate of biotin to SA being from 1 to 4.38. 37 Thus, a single target miRNA can attach probe DNA with several nanoprobes. Figure 1D exhibits the operation of an OECT in an electrolyte in the presence of the enzymatic reaction.…”

“…Au NPs have good conductivity and large surface area for binding electrochemical segments. Biotin on the nanoprobes can specially link to the streptavidin (SA) on the tail of the probe DNA with the binding rate of biotin to SA being from 1 to 4.38 . Thus, a single target miRNA can attach probe DNA with several nanoprobes.…”