We report the real-time detection of protein using SWNT-FET-based biosensors comprising DNA aptamers as molecular recognition elements. Anti-thrombin aptamers that are highly specific to serine protein thrombin were immobilized on the sidewall of a SWNT-FET using CDI-Tween linking molecules. The binding of thrombin aptamers to SWNT-FETs causes a rightward shift of the threshold gate voltages, presumably due to the negatively charged backbone of the DNA aptamers. While the addition of thrombin solution causes an abrupt decrease in the conductance of the thrombin aptamer immobilized SWNT-FET, no noticeable change was observed with elastase.

Recent advances in nanotechnology have enabled the development of nanoscale sensors that outperform conventional biosensors. This review summarizes the nanoscale biosensors that use aptamers as molecular recognition elements. The advantages of aptamers over antibodies as sensors are highlighted. These advantages are especially apparent with electrical sensors such as electrochemical sensors or those using field-effect transistors.

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The construction and screening of metagenomic libraries constitute a valuable resource for obtaining novel biocatalysts. In this work, we present the construction of a metagenomic library in Escherichia coli using fosmid and microbial DNA directly isolated from forest topsoil and screened for lipolytic enzymes. The library consisted of 33,700 clones with an average DNA insert size of 35 kb. Eight unique lipolytic active clones were obtained from the metagenomic library on the basis of tributyrin hydrolysis. Subsequently, secondary libraries in a high-copy-number plasmid were generated to select lipolytic subclones and to characterize the individual genes responsible for the lipolytic activity. DNA sequence analysis of six genes revealed that the enzymes encoded by the metagenomic genes for lipolytic activity were novel with 34-48% similarity to known enzymes. They had conserved sequences similar to those in the hormone-sensitive lipase family. Based on their deduced amino acid similarity, the six genes encoding lipolytic enzymes were further divided into three subgroups, the identities among which ranged from 33% to 45%. The six predicted gene products were successfully expressed in E. coli and secreted into the culture broth. Most of the secreted enzymes showed a catalytic activity for hydrolysis of p-nitrophenyl butyrate (C(4)) but not p-nitrophenyl palmitate (C(16)).

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