Stepwise amplification

The 67-fold amplification obtained for polymer 3 is restricted by an inherent limitation of the wired in series design. As the exciton travels in a one-dimensional random walk process down the polymer chain, it has equal opportunity to visit a preceding or an ensuing receptor. This represents 134 random stepwise movements for 134 phenylene ethynylene units, and so much of the receptor sampling by the exciton is redundant. Increasing the efficiency of receptor sampling requires maximization of the number of different receptors that an exciton can visit throughout its lifetime. To achieve this end we extended the polymer sensor into two dimensions by use of a thin film and thereby increased the sensitivity. 

The principle of in vitro selection is governed by a number of the same principles that apply to the Darwinian theory of evolution, as shown in Figure 2. First, the random sequence DNA is prepared by automated solid-phase synthesis. A mixture of four types of nucleotide is added in a stepwise condensation reaction process. When necessary, this DNA library may be converted to an RNA library by in vitro transcription or to a peptide library by in vitro translation. Second, the prepared DNA, RNA, or peptide library is subjected to affinity selection, and the molecules that bind to a target molecule are selected. Because only a very small part of the library is selected in each selection, the selected fraction is then amplified by a polymerase chain reaction (PCR) or a reverse transcription PCR (RT-PCR) technique. Successive selection and amplification cycles bring about an exponential increase in the abundance of the targeting DNA, RNA, or peptide until it dominates the population. 

Figure 9 Stepwise assembly of a DNA-sensing electrode by the functionalization of the conductive support with the sensing oligonucleotide (13), interaction with the target DNA (14) pre-hybridized with the biotinylated oligonucleotide (15), and the association of the avidin-HRP conjugate (16). The biocatalyzed precipitation of the insoluble product (8) is used as an amplification route for the electrochemical transduction of the biorecognition event.

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