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Carbon Nanotube Arrays for Bacteria

Liu, Haiying
Michigan Technological University
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Our specific goal is to develop novel electrochemical biosensors for foodborne bacteria by functionalizing vertically aligned carbon nanotubes arrays with redox-active carbohydrate conjugates. Since biological and electrical activities are linked by redox-active carbohydrate conjugates, the biosensors will effectively transduce biological activities of carbohydrate-protein interactions into electrical activities at carbon nanotube surfaces, which can be monitored by electrochemical technology such as cyclic voltammetry and differential pulse voltammetry.

The specific objectives are designed to achieve electrochemical monitoring of pathogen recognition:

  1. Design and synthesize amino-functionalized redox-active carbohydrate conjugates. The redox-active moiety will be conjugated closely to carbohydrates so that environmental changes around the redox-active moiety caused by biological recognition can result in changes of its electrochemical properties such as potential and current. We will design, synthesize and use amino-functionalized redox-active carbohydrate conjugates to functionalize vertically aligned carbon nanotubes arrays to enhance sensitivity of electrochemical monitoring. These redox-active moieties conjugated to carbohydrates will include ferrocene and tetrathiafulvalene, and the carbohydrate moiety will be varied to study different carbohydrate-protein interactions including pathogen recognition involving foodborne Escherichia coli.
  2. Construct biosensors to monitor pathogen recognition involving foodborne Escherichia coli by functionalizing cap ends of vertically aligned carbon nanotube arrays with amino-functionalised redox-active carbohydrate conjugates. Combination of redox-active carbohydrate conjugates with vertically aligned CNT arrays is expected to significantly enhance the sensitivity of electrochemical monitoring of pathogen recognition.
  3. Monitor enzymatic glycosylation for potential useful information about how enzymes bind to substrates.
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NON-TECHNICAL SUMMARY: Problem: Pathogenic organisms such as E. coli threaten our food and water safety. Such a threat could be substantially mitigated if pathogens are detected at an early stage. Current methods to monitor pathogens require complicated sample purification procedures, exogenous labels, labile reagents, or expensive, heavy, power-hungry instrumentation. The purpose of the project: We plan to develop novel electrochemical biosensors for food-borne bacteria by attaching redox-active carbohydrate conjugates to carbon nanotubes. The proposed research will result in new biosensors to in situ monitor pathogens by electrochemical technology without complicated sample purification procedures.

APPROACH: In this proposed research, we will develop a new, general, real-time, quick, operationally simple, sensitive and specific methodology to monitor carbohydrate-protein interactions by designing interfaces that detect biological and electrical signals in both directions across the interface. The biological and electrical activities are linked by redox-active carbohydrate conjugates that can transduce biological activities into electrical signals. The redox-active species conjugated to carbohydrates will include ferrocene and tetrathiafulvalene. This new methodology is based on a hypothesis that electrochemical properties (potential and current) of the redox moieties will change due to environmental changes around the redox- active species when proteins or cells bind redox-active carbohydrates immobilized on a surface, which can be monitored by electrochemical technology such as cyclic voltammetry and differential pulse voltammetry. The research will be carried as follows: -- Synthesize amino-functionalized redox-active carbohydrate conjugates -- Functionalize vertically aligned carbon nanotube arrays with the redox-active carbohydrate conjugates with controlled density to optimize carbohydrate-protein interactions -- Electrochemically monitor carbohydrate-protein interactions involving enzymatic glycosylation and pathogen recognition.

PROGRESS: 2007/01 TO 2007/12
OUTPUTS: 1) We have succeeded in growing vertically-aligned carbon nanotubes (CNTs) on conducting Si substrates. These CNTs are designated to use as the nanoscale electrodes for redox-active carbohydrate conjugates that will recognize pili of bacteria. In order to enhance the signal-to-noise level, only the tips of these CNTs will be exposed and functionalized to bind with the redox-active carbohydrates. To achieve this goal, we need to shield the conducing substrates and CNTs with insulators. 2) We have succeeded in shielding the CNTs and the substrate while maintaining the vertical alignment of the CNTs. This has been a challenging task. We finally succeeded in using diluted PMMA photoresist / polymer to embed the entire array of vertically-align CNTs. 3) We have succeeded to expose the tips of the embedded by mechanical polishing. Since the samples are coated with insulator, charging effect under electron microscopy has made the identification of these exposed CNT tips extremely difficult. We finally succeeded by combining with micro-Raman spectroscopy. 4) We have succeeded in connecting the conducting substrate with a shielded wire. The entire device was then fully shielded with insulator so that only the CNT tips will expose for the electrochemical testing. 5) We have successfully developed pre-polymerization and post-polymerization functionalization approaches to fast prepare well-defined conjugated glycopolymers for functionalization of CNTs. We have successfully demonstrated significantly enhanced interactions of water-soluble conjugated glycopolymers such as glyco(p-phenylene)s and fluorene-based conjugated glycopolymers with Escherichia coli (E. coli), through multivalent cooperative interactions, forming highly fluorescent bacteria clusters, which can easily observed by naked eye or fluorescent microscope. 6) We have successfully prepared thiol-functionalized ferrocene-mannose conjugate, thiol-functionalized viologen-mannose conjugate, and a series of thiol- and amine-functionalized tetrathiafulvalene-carbohydrate conjugates. 7) We have successfully used electrodes modified with self-assembled monolayers (SAMs) of ferrocene-mannose conjugate and HO(CH2)6SH to selectively detect E. coli. We chose two E. coli strains ORN178 and ORN208 for testing and control experiments as the ORN178 strain expresses the wild-type type 1 pili that selectively bind to mannose, whereas the ORN208 strain is deficient of the fimH gene and expresses abnormal type 1 pili that fail to mediate specific binding to alfa-mannose. The presence of 400 cells of ORN178 E. coli causes a significant positive shift (about 40 mV) of redox potential of ferrocene from SAMs and a decrease of oxidation or reduction current of the ferrocene. However, the presence of 400 cells of mutant ORN208 E. coli doesn't change electrochemical properties of the ferrocene from SAMs since mutant E. coli fails to bind to mannose moieties from the SAMs.
PARTICIPANTS: Haiying Liu, Assistant professsor, Department of Chemistry, Michigan Tech. Yoke Khin Yap, Associate Professor, Department of Physics, Michigan Tech Pushpalatha Murthy, Professor, Department of Chemistry, Michigan Tech Martin Thompson, Assistnat professor, Department of Chemistry, Michigan Tech Archana Pandey, Ph.D. student, Department of Physics, Michigan Tech Venkat R. Donuru, Ph.D. student, Department of Chemistry, Michigan Tech Singaravelu Velayudham, Ph.D. student, Department of Chemistry, Michigan Tech Cuihua Xue, Postdoctoral associate, Department of Chemistry, Michigan Tech
TARGET AUDIENCES: None at this time

IMPACT: 2007/01 TO 2007/12
1) The fabrication of CNT arrays for high signal-to-noise electrochemical detection is challenging. Previously reported works were limited to the use of low density CNTs so that effective shielding with insulator can be achieved. These will required the growth of CNTs on patterned catalyst arrays created by electron beam lithography or other treatments. In contrast, we have succeeded in fabricating CNT arrays by as grown samples. The density of our CNTs is at least one order of magnitude higher.
2) The use of high-density CNTs in this project has made the shielding process challenging. We found that diluted PMMA with optimum concentration enable the penetration of the insulator to fill up all the spacing between CNTs. PMMA has posted the advantage for further photolithography process for advanced devices in the future.
3) Because of the relatively weak mechanical property of PMMA, exposing the tips of CNTs without breaking the insulating layer has been challenging. We finally succeeded to identify the optimum procedure to do so and will contribute to future device fabrication.
4) The combined use of scanning electron microscopy and Raman microscopy has help to identify the tips of CNTs. Other reported works did not sure such evidence.
5) Preparation of water-soluble well-defined conjugated polymers is very challenging through post-polymerization functionalization due to incomplete reactions. We have developed post-polymerization functionalization approach to quickly prepare well-defined fluorescent conjugated glycopolymers such as glycopoly(p-phenylene)s, fluorene-based conjugated glycopolymers and glycopolythiophenes through thioether bridges. We have successfully use oligo(ethylene glycol) and poly(ethylene glycol) as tethered spacers to make conjugated glycopolymers highly soluble in aqueous solutions, and prevent non-specific interactions. This post-polymerization functionalization approach will offer a fast and cost-effective means to prepare well-defined conjugated glycopolymers for bacterial biosensing applications.
6) Synthesis of thiol-functionalized and amine-functionalized redox-active carbohydrate conjugates are extremely challenging due to multi-step organic reactions and purifications. Successful preparation of these redox-active carbohydrates will facilitate bacterial biosensing applications by using electrochemical technology.
7) We have successfully developed a new electrochemical approach to selectively detect E. coli by monitoring carbohydrate-bacterium interactions. Binding of bacterial pili to carbohydrates from monolayers of ferrocene-mannose conjugate causes changes of electrochemical properties of ferrocene moieties such as potential and current. The new methodology will be easily extended to monitor other carbohydrate-protein interactions involving other pathogenic bacteria and influenza virus by using other carbohydrates instead of mannose.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Escherichia coli
Prevention and Control