ADVANCED DIAGNOSTICS TECHNOLOGIES

Micro/nano optical and electrochemical diagnostic systems for biodefense and emerging infections

Yong Chen, University of California, Los Angeles

Abstract:
The objective of the sub-project is to develop and provide practical and effective systems for detecting various biological agents in support of Pacific Southwest RCE’s overall efforts in biodefense and infectious disease.

Current diagnostic tools for detecting infectious agents rely heavily on serum antibody expression, polymerase chain reaction (PCR), and/or culture. Although reliable, these methods all require substantial time, extensive human involvement, laboratory-based equipment, and relatively high pathogen loads. The significance of such limitations is clearly illustrated by the initial efforts to detect the delivery of anthrax-laced letters in September, 2001 in Florida and Washington, D.C., and identify and quarantine patients with severe acute respiratory syndrome (SARS) in China in 2003. In countering bioterrorism and emerging infectious diseases effectively, we believe that one of the major challenges is rapid and reliable detection of the nature and breadth of an infectious event. To meet this challenge, this sub-project will focus on the development of micro/nano optical and electrochemical diagnostic systems in the Region IX RCE.

Recent research progress in the areas of microelectromechanical system (MEMS), nanotechnology and biosensors has provided revolutionary pathways for solving the aforementioned problems. For example, as demonstrated in our laboratories, MEMS micro fluidic technology has enabled the creation of so-called lab-on-a-chip (LOC) portable biodetection systems (Kim) , in which microfluidic operations are integrated “on chip” free of pumps, valves and channels for automated sample preparation. For a detection process, we have pioneered advanced optical biosensing technology—fluorescence correlation spectroscopy (FCS) (Ho)— which has pushed the DNA detection limit down to the single-molecule level. Additionally, the highly sensitive electrochemical enzyme-multiplied immunoassay technique (ElectroEMIT by Monbouquette) can amplify detection signals in the form of an electrical current for digitized recording. Coupling ElectroEMIT with nanoscale electronic circuits (Chen) promises significant improvements in the signal/noise ratio, sensitivity and density of sensor arrays. By integrating these Bio/MEMS/Nano techniques, we envision various miniaturized portable biodetection platforms to detect specific pathogens and pathogen-related biomarkers such as proteins, antibodies, RNA and DNA for multiple infectious agents with ultrahigh sensitivities and specificities.

 

Microfluidics-HPLC-MS for early diagnostics

Yu-Chong Tai, California Institute of Technology

Abstract:
Mass spectrometry (MS) is one of the most sensitive and yet generic tool for biochemical detection. In addition, the feasibility (in fact, the best success) of using MS for military biological agent (e.g., anthrax) detection has been recently demonstrated. However, the advancement is limited that even the most advanced diagnostic MS system is vehicle-mounted and for airborne pathogens only ( http://www.ornl.gov/info/ornlreview/v33_3_00/mass.htm ).

Here, we propose to use chip-based HPLC-MS for early pathogen diagnostics for a host and our proposal has three major specific aims:

1. The first aim is the early diagnostics of host pathogens. The sample preparation is far more complicated for host infection by pathogens and for airborne pathogens.
2. The second aim is to use microfluidics to simplify and automate the sample preparations before the MS detection. The most important advantage of this approach is to improve the sensitivity of the overall system, which in term improves the capability of early detection. The other advantage is then the automation and miniaturization.
3. The third aim is to develop a truly portable tool by replacing the mass spectrometer (during the assay phase) with other detection methods such as electrochemistry and electroluminescence.

This work is a collaboration with Terry Lee and Markus Kalkum at City of Hope. The expertise that our team has in the areas of immunology, microbiology, mass spectrometry, and micro/nanofluidics technology provides a valuable component to develop new approaches to early pathogen detection, interact with other RCE cores and projects, and translate research results into effective analytical systems and methods.

Scope of Work: Microfluidic platform for the detection of Botulinum toxin

Markus Kalkum, (Tai - subcontract), City of Hope

Abstract:
The goal of this project is to develop a portable device capable of rapidly (< 45 min) detecting botulinum neurotoxins (BoNTs) in serum at levels lower than the best available commercial assays (80 pg/ml (4) ). The required sensitivity will by achieved by affinity capture of the BoNT protein complex using immobilized antibodies followed by detection of the specific enzymatic activity of the toxin by cleavage of reporter peptide. The reporter peptide would utilize the IQ protease assay technology commercialized by Pierce. To avoid the complications of working directly with active BoNT, these first year feasibility studies will use metalloproteinases (such as MMP-3), which like BoNT utilize zinc in their catalytic site. Accordingly, the following specific aims are proposed for the first year feasibility studies.

1. Demonstrate the affinity capture of MMP-3 from serum into a microfluidic device.
2. Demonstrate a high sensitivity fluorescent assay based on the MMP-3 immobilization .

Patterns of host response to systemic infectious disease

David Relman, Stanford University

Abstract:
Early recognition of infectious disease is critical in any successful strategy for prevention and management, especially in the setting of bioterrorism. Unfortunately, routine diagnostic and prognostic capabilities in infectious diseases are suboptimal, especially at the early phases of infection or illness. Recent exploratory efforts to examine host responses to infection using genome-wide methods have provided evidence of host discrimination among different microbial agents. Motivated by these needs and findings, we propose the following aims: Aim 1. To analyze host response patterns in non-human primates with smallpox and monkeypox, as well as humans with naturally-acquired monkeypox in the Democratic Republic of Congo (DRC). We will compare and contrast host responses in NHP with smallpox versus monkeypox, and test the applicability of these findings to humans with monkeypox, using blood subsets and other tissues, a state-of-the-art comprehensive human genome oligonucleotide DNA microarray, and a suite of pattern recognition algorithms including data modeling approaches. Aim 2. To analyze host response patterns in NHP infected with Ebola (Zaire) and Marburg viruses. The differential features of these related disease processes will be explored, using methods similar those of the previous Aim, ex vivo cell subset separation will allow identification of cell type-specific response patterns. Aim 3. To examine genome-wide responses of humans with undifferentiated fever at an early stage of systemic disease caused by a variety of microbial pathogens, including category B and C agents. A microbiological diagnosis will be ascertained in patients from a study site in Laos using routine methods. Host response patterns from Laos will be compared to patterns previously collected from patients with similar diagnoses in Ho Chi Minh City, Vietnam and Kathmandu, Nepal. The broad, long-term objectives of this work are to develop rules and signatures for diagnosis and prognosis of systemic infectious disease, based on patterns of host response. This work has public health relevance because it will lead to more effective planning and clinical management of infectious diseases cases as a result of more timely diagnosis and prediction of clinical course.