Surface immobilizable chelator for label-free electrical detection of pyrophosphatew

Nucleotide incorporation reactions, catalyzed by DNA and RNA polymerases, are critically important in the biological processes of living systems. Their common byproduct, pyrophosphate (PPi), is a negatively charged small molecule typically detected using optical techniques, such as chemiluminescence. Label-free electrical monitoring of biochemical reactions offers several advantages including increased portability and improved integration. The former advantage is due to the elimination of bulky optical measurement components and the latter is due to the ability to fabricate many individually addressable electronic devices at microor nanoscale. Scalable semiconductor manufacturing techniques can be adapted to produce dense, highly reproducible sensor arrays to process samples and signals in a highly parallel fashion. We are developing an electrical signal detection platform capable of detecting DNA synthesis reactions by making use of the intrinsic physicochemical properties of PPi. Here we report the synthesis of a PPi-selective receptor, its surface immobilization and application to label-free electrical detection on a field-effect transistor (FET) device. Various optical PPi detection technologies have been developed. Among these, luciferase-based PPi detection has been used for bacterial detection and DNA sequencing applications. Non-enzymatic PPi detection technologies have also been reported. These include fluorescenceand absorption-based detection using PPi chelators, which can detect submicromolar PPi in bulk solution. One class of chelators is designed such that they can bind to indicator dyes, where PPi is detected either colorimetrically or fluorescently when dye molecules are displaced from chelator by PPi binding. Extending chelation-based sensing to surface-sensitive electrical detection requires a chelator compatible with surface immobilization and selective to the target analyte. Surface capture of PPi signaling molecules is expected to enhance the sensitivity of field-effect devices to PPi in a process that we call ‘‘signal immobilization’’. The negatively-charged PPi molecules are expected to decrease the number of positively charged carriers in a p-type field-effect transistor (FET) sensor functionalized with such a chelator, resulting in a decrease in threshold voltage. In order to test this ‘‘signal immobilization’’ concept, we designed a new chelator with three functional components: a binding site, a linker, and a handle. The binding site selectively captures PPi from solution, while the linker between the binding site and handle provides steric flexibility. Finally, the handle ensures that the chelator can be selectively attached to a chemically compatible surface. The selected PPi chelator was based on di-(2-picolyl) amine (DPA), which has demonstrated strong binding affinity to PPi and is relatively straightforward to synthesize. The hydroxyl groups of 5-nitro-1,3-bishydroxymethylbenzene were first tosylated to accelerate substitution with DPA (Scheme 1). After DPA substitution, the nitro group was reduced to an amine by catalytic hydrogenation. The addition of zinc nitrate produces a functional complex with two Zn coordination sites per chelator molecule. Basic functionality of the synthesized chelator was verified in solution. Selective binding studies were performed using a coumarin-based fluorescent dye or a colorimetric dye, pyrocatechol violet (PV). In the case of the fluorescent dye,