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Chapter 9
Gold Nanoparticle Based Surface Energy Transfer Probe for Accurate Identification of Biological Agents DNA Paresh Chandra Ray*, Gopala Krishna Darbha, Oleg Tovmachenko, Uma Shanker Rai , Jelani Griffin, William Hardy and Ana Balarezo Department of Chemistry, Jackson State University, Jackson, MS, USA
Rapid differentiation and accurate identification of bioagents are crucial to planning timely and appropriate measures for public safety. Driven by the need to detect bioagents selectively, the development of a miniaturized, inexpensive and battery operated ultra-sensitive gold nanoparticle-based surface energy transfer (NSET) probe for screening of the bioagents DNA with excellent sensitivity (800 femto-molar) and selectivity (single base pair mismatch) is reported here. In the presence of various target sequences, detection of sequence specific DNA is possible via independent hybridization process. As proof of concept, multiplexed detection of two target sequences, 1) oligonucleotide sequence associated with the anthrax lethal factor and 2) sequence related to positions 1027–1057 of the E.coli DNA which codes for the 23S rRNA, have been demonstrated with high sensitivity and specificity. The quenching efficiency as a function of distance is investigated. The mechanism of distant dependent fluorescence quenching has been discussed.
© 2009 American Chemical Society
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In Nanoscience and Nanotechnology for Chemical and Biological Defense; Nagarajan, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.
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Introduction The biological weapon has been around at least since the Middle Ages when soldiers catapulted the bodies of dead smallpox victims over fortress walls in the hope of infecting their enemies or at least demoralizing them. Lately, biological agents have been appearing in the news with increasing frequency. Biological agents are of concern because of the lag time between a biological attack and the appearance of symptoms in those exposed, biological weapons could be devastating. Many biological agents are contagious, and during this lag time, infected persons could continue to spread the disease, further increasing its reach. As a result, the availability of rapid, sensitive and cost-effective diagnostic methods is paramount to the success of a comprehensive national health security system in the USA (1,2) . Ideally, detection systems should be capable of rapidly detecting and confirming biothreat agents, including modified or previously uncharacterized agents, directly from complex matrix samples, with no false results. Furthermore, the instrument should be portable, userfriendly, and capable of testing for multiple agents simultaneously. Many local and state authorities are inadequately prepared to deal with biological-based incidents, and first responders to such incidents will face considerable risk. In addition, since public health personnel rarely encounter any of the 30 or so pathogens on various agency threat lists, the ability to rapidly identify their infection is waning. So sensors must not only be sensitive and specific, but must also be able to accurately detect a variety of pathogens. Driven by the need, current efforts have been focused on the development of miniaturized, inexpensive and battery operated ultra-sensitive gold nanoparticle based surface energy transfer (NSET) probe for screening bioagents DNA which has excellent sensitivity and selectivity. Conventional analytical methods (3-5) used to detect biological agents, such as high-performance liquid chromatography, gas chromatography, and mass spectroscopy, require expensive equipments that may be difficult to fielddeploy. Standard microbiological methods (6,7), such as culturing and microscopic examination, are time-consuming and labor-intensive. The increasing availability of nanostructures with highly controlled optical properties in the nanometer size range has created widespread interest in their use in biotechnological system for diagnostic application and biological imaging (833). Merging biotechnology with nano science will allow us not only to take advantage of the improved evolutionary biological components to generate new smart sensors but also to apply today's advanced characterization and fabrication techniques to solve biodefense problems. Although still in its infancy, the application of surface-functionalized nanomaterials such as nanoparticles in sequence recognition schemes has shown (8-34) great promise in achieving high sensitivity and specificity, which are difficult to achieve by conventional methods. In this chapter, we have discussed our recent research on gold nanoparticle (Au/NP) based fluorescence resonance energy transfer (FRET) assay to detect multiple bioagenst DNA together. We also described the fabrication of a miniaturized, inexpensive and battery operated ultra-sensitive laser-induced fluorescence (LIF) optical fiber sensor, based on quenching of the LIF signal by gold nanoparticle, to detect single base-mismatch DNA.
In Nanoscience and Nanotechnology for Chemical and Biological Defense; Nagarajan, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on August 28, 2015 | http://pubs.acs.org Publication Date: December 24, 2009 | doi: 10.1021/bk-2009-1016.ch009
117 Recently, there have been many reports for the development of fluorescence-based assays for bioagents DNA detection (35-37). These assays are based on Föster resonance energy transfer (FRET) (38) or non-FRET quenching mechanisms. Although FRET technology is very convenient and can be applied routinely at the single molecule detection limit, the length scale for the detection using FRET-based method is limited by the nature of the dipoledipole mechanism, which effectively constrains the length scales to distances on the order of
