There is national interest in the development of sophisticated materials that can automatically detect and respond to chemical and biological threats without the need for human intervention. In living systems, cell membranes perform such functions on a routine basis, detecting threats, communicating with the cell, and triggering automatic responses such as the opening and closing of ion channels. The purpose of this project was to learn how to replicate simple threat detection and response functions within artificial membrane systems. The original goals toward developing 'smart skin' assemblies included: (1) synthesizing functionalized nanoparticles to produce electrochemically responsive systems within a lipid bilayer host matrices, (2) calculating the energetics of nanoparticle-lipid interactions and pore formation, and (3) determining the mechanism of insertion of nanoparticles in lipid bilayers via imaging and electrochemistry. There are a few reports of the use of programmable materials to open and close pores in rigid hosts such as mesoporous materials using either heat or light activation. However, none of these materials can regulate themselves in response to the detection of threats. The strategies we investigated in this project involve learning how to use programmable nanomaterials to automatically eliminate open channels within a lipid bilayer host when 'threats' are detected. We generated and characterized functionalized nanoparticles that can be used to create synthetic pores through the membrane and investigated methods of eliminating the pores either through electrochemistry, change in pH, etc. We also focused on characterizing the behavior of functionalized gold NPs in different lipid membranes and lipid vesicles and coupled these results to modeling efforts designed to gain an understanding of the interaction of nanoparticles within lipid assemblies.
The excitement surrounding the marriage of biosensors and nanotechnology is palpable even from a cursory examination of the scientific literature. Indeed, the word “nano” might be in danger of being overused and reduced to a cliché, although probably essential for publishing papers or securing research funding. The biosensor literature is littered with clever or catchy acronyms, birds being apparently favored (“CANARY”, “SPARROW”), quite apart from “electronic tongue,” “electronic nose,” and so on. Although biosensors have been around since glucose monitors were commercialized in the 1970s, the transition of laboratory research and innumerable research papers on biosensors into the world of commerce has lagged. There are several reasons for this phenomenon including the infamous “valley of death” afflicting entrepreneurs emerging from academic environment into the industrial world, where the rules for success can be radically different. In this context, musings on biosensors and especially nanobiosensors in an open access journal such as Journal of Biosensors and Bioelectronics is topical and appropriate especially since market surveys of biosensors are prohibitively expensive, sometimes running into thousands of dollars for a single copy. The contents and predictions of market share for biosensors in these reports also keep changing every time a report is published. Not only that, the market share projections for biosensors differs considerably amongst various reports. An editorial provides the opportunity to offer personal opinions and perhaps stimulate debate on a particular topic. In this sense, editorials are a departure from the rigor of a research paper. This editorial is no exception. With this preamble, it is worthwhile to stop and ponder the status of commercial biosensors and nanobiosensors.
I used supramolecular self-assembling cyanine and the polyamine spermine binding to Escherichia coli genomic DNA as a model for DNA collapse during high throughput screening. Polyamine binding to DNA converts the normally right handed B-DNA into left handed Z-DNA conformation. Polyamine binding to DNA was inhibited by the supramolecular self-assembling cyanine. Self-assembly of cyanine upon DNA scaffold was likewise competitively inhibited by spermine as signaled by fluorescence quench from DNA-cyanine ensemble. Sequence of DNA exposure to cyanine or spermine was critical in determining the magnitude of fluorescence quench. Methanol potentiated spermine inhibition by >10-fold. The IC{sub 50} for spermine inhibition was 0.35 {+-} 0.03 {micro}M and the association constant Ka was 2.86 x 10{sup -6}M. Reversibility of the DNA-polyamine interactions was evident from quench mitigation at higher concentrations of cyanine. System flexibility was demonstrated by similar spermine interactions with {lambda}DNA. The choices and rationale regarding the polyamine, the cyanine dye as well as the remarkable effects of methanol are discussed in detail. Cyanine might be a safer alternative to the mutagenic toxin ethidium bromide for investigating DNA-drug interactions. The combined actions of polyamines and alcohols mediate DNA collapse producing hybrid bio-nanomaterials with novel signaling properties that might be useful in biosensor applications. Finally, this work will be submitted to Analytical Sciences (Japan) for publication. This journal published our earlier, related work on cyanine supramolecular self-assembly upon a variety of nucleic acid scaffolds.
ElectroNeedles technology was developed as part of an earlier Grand Challenge effort on Bio-Micro Fuel Cell project. During this earlier work, the fabrication of the ElectroNeedles was accomplished along with proof-of-concept work on several electrochemically active analytes such as glucose, quinone and ferricyanide. Additionally, earlier work demonstrated technology potential in the field of immunosensors by specifically detecting Troponin, a cardiac biomarker. The current work focused upon fabrication process reproducibility of the ElectroNeedles and then using the devices to sensitively detect p-cresol, a biomarker for kidney failure or nephrotoxicity. Valuable lessons were learned regarding fabrication assurance and quality. The detection of p-cresol was accomplished by electrochemistry as well as using fluorescence to benchmark ElectroNeedles performance. Results from these studies will serve as a guide for the future fabrication processes involving ElectroNeedles as well as provide the groundwork necessary to expand technology applications. One paper has been accepted for publication acknowledging LDRD funding (K. E. Achyuthan et al, Comb. Chem. & HTS, 2008). We are exploring the scope for a second paper describing the applications potential of this technology.
Columbia University has developed a sensitive highly multiplexed system for genetic identification of nucleic acid targets. The primary obstacle to implementing this technology is the high rate of false positives due to high levels of unbound reporters that remain within the system after hybridization. The ability to distinguish between free reporters and reporters bound to targets limits the use of this technology. We previously demonstrated a new electrokinetic method for binary separation of kb pair long DNA molecules and oligonucleotides. The purpose of this project 99864 is to take these previous demonstrations and further develop the technique and hardware for field use. Specifically, our objective was to implement separation in a heterogeneous sample (containing target DNA and background oligo), to perform the separation in a flow-based device, and to develop all of the components necessary for field testing a breadboard prototype system.