Item Details

abstract

Aptamer-based screening for illicit drugs represents an attractive alternative to traditional ELISA-based methods due to lower cost and greater reagent stability and specificity offered by single stranded DNA (ssDNA) aptamers relative to their antibody counterparts. Aptamer-based screening requires both effective detection (via fluorescence emission) and identification of aptamer sequences uniquely qualified to select for the desired target. This work focuses on the use of nitrogen-doped carbon dots (NCDs) to facilitate selective detection of the aptamer-bound drug target, such as cocaine.Carbon dots (CDs) are fluorescent nanomaterials that have potential for application in biosensing and medical diagnostics due to their biocompatibility and low toxicity. This study involves two types of carbon dots synthesized in-house and characterized for their physicochemical properties: unmodified CDs from citric acid, and nitrogen-doped (ethylenediamine modified) NCDs. Scanning electron microscopy showed the NCDs are smaller in size (2-9 nm) than the unmodified CDs (6-12 nm). Also, fluorescence emission studies revealed that the NCDs are more highly fluorescent than unmodified CDs. These studies reveal that ssDNA will associate with NCDs and by so doing, will experience a significant change in net electrophoretic mobility relative to free DNA. In addition to nonsense DNA and randomer DNA samples, we employed a known cocaine aptamer sequence consisting of 32 nucleotides, covalently labeled with carboxyfluorescein (6-FAM). The aptamer fluorescence is quenched upon binding to the NCDs. However, the aptamer signal is restored upon addition of the target, cocaine, thus allowing for the drug’s selective detection due to preferential binding of the ssDNA with its target compared to binding with NCDs. It is anticipated that this NCD-mediated competitive binding can be exploited for the capillary transient isotachophoresis (ctITP) separation of drug target-bound DNA sequences from free DNA libraries with a view to discovering aptamers against illicit drugs. Another application of NCDs is revealed, which permits for the separation of ssDNA based on size. The NCDs were used as buffer additives in ctITP with laser induced fluorescence (LIF) detection, which enabled the separation of FAM-labeled ssDNA molecules ranging from 32-100 bases in length.

subject

Aptamer

Capillary electrophoresis

Carbon dots

Separation

Small molecule

contributor

Roy, Debashish (author)

Colyer, Christa L (committee chair)

Marrs, Glen S (committee member)

Geyer, Scott M (committee member)

Hinze, Willie L (committee member)

King, Bruce (committee member)

date

2021-09-01T08:35:26Z (accessioned)

2021 (issued)

degree

Chemistry (discipline)

2022-08-31 (liftdate)

embargo

2022-08-31 (terms)

identifier

http://hdl.handle.net/10339/99061 (uri)

language

en (iso)

publisher

Wake Forest University

title

CARBON DOTS AS AIDS FOR ssDNA SEPARATION AND APTAMER DISCOVERY AGAINST SMALL MOLECULE DRUGS BY CAPILLARY ELECTROPHORESIS

type

Dissertation