Enhancement of Electrochemical Signal on Gold Electrodes by Polyvalent Esterase−Dendrimer Clusters

Abstract

5'-Maleimide-oligodeoxynucleotide was conjugated with single sulfhydryl group of cystamine core poly(amidoamine) dendrimers of different generations. Amino groups on the dendrimer moiety were modified with maleimide and coupled to the cysteine 118 of esterase 2 from Alicyclobacillus acidocaldarius in a site-specific manner. Polyvalent esterase-dendrimer-oligodeoxynucleotide clusters were hybridized to capture oligodeoxynucleotides immobilized on a gold electrode. The amperometric signal of p-aminophenol was detected following the esterase-catalyzed hydrolysis of p-aminophenylbutyrate. The multiple anchoring of the esterase reporter via generation 3-and generation 5-derived clusters exhibited 10- and 100-fold signal enhancement, respectively, as compared to monovalent esterase-oligonucleotide conjugate. The polyvalent and monovalent reporters were comparable in their abilities regarding mismatch discrimination.

Detection of bacterial 16S rRNA using multivalent dendrimer-reporter enzyme conjugates

“…Similar experiments were performed with G5 polyamidoamine cluster, which possesses a higher number of EST2 molecules compared to G3 cluster (Humenik et al, 2008). As previously described (Humenik et al, 2008), higher unspecific binding onto blank gold electrode as compared to G3 EST2-dendrimer-ODN cluster was observed, but no further improvement of detection limit for E. coli could be achieved (data not shown).…”

“…Obviously, larger hydrodynamic radius of EST2-dendrimer-ODN cluster handicapped hybridization to dense anchored RNA at high analyte concentration (10 6 -10 8 cfu). At low analyte concentrations (below 10 4 cfu) there are sufficient hybridization possibilities without steric hindrance (Humenik et al, 2008) allowing multiple binding of the reporter enzyme per hybridization event and production of a significant signal enhancement. Due to the high hydrodynamic radius of EST2-dendrimer-ODN cluster the linear region became narrower in comparison to EST2-ODN conjugate.…”

“…p-Aminophenyl butyrate (pAPB) and monovalent EST2-ODN conjugate, with universal detector sequence UD1082, complementary to conserved region of 16S rRNA starting from position 1082 of Escherichia coli K12 16S rRNA sequence, were prepared as described . Multivalent EST2-dendrimer-UD1082 conjugates, derived from G3 and G5 of PAMAM cystamine core dendrimer, were prepared by described procedures (Tomalia et al, 2003;Humenik et al, 2008). The ratio of EST2 to ODN was 7:1 for G3 and 10:1 for G5 cluster.…”

“…This construct provided a significant signal enhancement of DNA/DNA hybridization as compared to simple EST2-ODN conjugates (Humenik et al, 2008). In the present work we applied EST2-dendrimer-ODN clusters for identification of microorganisms via rRNA/DNA hybridization of bacterial 16S rRNA.…”

Electrochemical Detection of RNA

“…This biosensor achieved the detection of 500 cfu E. coli within less than 40 min and was further improved by establishing a rapid one-step lysis method allowing sample preparation suitable for an on-site application (unpublished results). Additionally, signal amplification was achieved by using multiple EST2 molecules conjugated via polyamidoamine dendrimer to single universal detector oligodeoxynucleotide (Humenik et al 2008). This resulted in a detection limit of 50 cfu E. coli per electrode sensor within less than 40 min (P€ ohlmann et al 2009b).…”