DETECTION, IDENTIFICATION, AND MONITORING

Publication Details

Postgenomic Technologies – Andrew Pitt, University of Glasgow, UK

Dr. Andrew Pitt opened the session by providing an overview of the state of technology in genomic analysis. Dr. Pitt introduced the topic by describing three different eras of technology development, the genomic, the post-genomic, and the pre-post genomic. During the genomic era, the development of early sequencing technology provided researchers with the tool to understand the genomic underpinnings of biology. However, early technology was slow and expensive, and these genomic analyses identified some of the limitations of genomics in predicting biological outcome. With the development of tools better able to study proteins, metabolites, and the like, science entered the post-genomic era. Today, Dr. Pitt argued, further advances in analytical technology have allowed for a new level of analysis and have, to some extent, returned us to what he called the pre-post-genomic era.

Recent years have seen major advances in the ability to sequence genes quickly and at lower cost, which allows for high throughput analyses. Today it costs approximately $5000 to sequence a genome, and it is believed that the $1000 genome is on the horizon (see discussion in footnote 6). Dr. Pitt noted that, to date, about 6,500 genomes have been sequenced worldwide which is creating a new challenge—massive amounts of data that we do not yet have the tools to fully analyze.

Beyond sequencing technology, Dr. Pitt noted that within the past five years there have been advances in the ability to do fine analysis of cellular environments. This degree of control allows for analysis of whole cells and of partial cells and has improved both the understanding of cellular components and mechanisms and the understanding of the linkage between the genome sequence and biological outcome.

Looking ahead to the next five years, Dr. Pitt predicted that significant advances would be made towards personalized medicine. Anticipated technological developments to support this include the development of multiplex protein arrays and improved analysis methods. There will be a greater focus on identification and study of biomarkers, and other technological developments will be targeted towards understanding RNA interference (RNAi) and the function of small interfering RNA (siRNA). There will also be an increased ability to perform molecular and physical observations of the role genetics play in organisms. Dr. Pitt noted that these developments are relevant to both Articles I and II of the BWC, with potential applications for detection, identification, and verification.

Bioforensics – Randall Murch, Virginia Polytechnic Institute and State University, USA

Dr. Randall Murch introduced the topic of bioforensics by noting his extensive experience in the area, both as a criminal investigator and as a forensic scientist. He described how, over the course of his career, the role of forensics has shifted from one of last-ditch effort on the part of the investigators to a position of prominence, where the forensic laboratory is brought in early in the process and assists throughout an investigation. Today, if a biological event occurred, forensics would be seen by investigators and leaders as a tool to help answer critical questions such as: what is or was released, who is responsible, where did it come from, what can we know with confidence, and by when?

Dr. Murch noted that in order to understand the role that bioforensics can play in answering these questions, it is important that certain terms be clearly defined. Forensic science is “the application of science in the investigation of legal and policy matters. It consists of analysis and interpretation of physical evidence to determine relevance to events, people, places, tools, methods, processes, intentions, [and] plans.” The overall goal of forensic science is to identify and characterize that evidence to aid and direct investigators as they seek to assign attribution, often by narrowing potential sources for the physical evidence. Attribution is “the assignment of a sample of questioned origin to a source of known origin to a high degree of scientific certainty.” When identifying samples and assigning attribution, Dr. Murch noted that it is critical that analysts have a sample of known origin with which to compare an evidentiary sample and that consistent, systematic, and validated methods be used for the analysis. The science often fills a special investigative role, and the results of a forensic analysis can feed into a broader analysis of all evidence available (from intelligence, investigative information, etc.).

In recent years, the emphasis on forensics within an investigation has grown, and as a result, the expectations and requirements for quality and reliability are increasing. Along with these rising expectations is increased scrutiny on the science used within the judicial system. In 2009, the U.S. National Research Council issued a report stating that forensic science and its performers have been shown or assessed to have significant gaps, shortcomings, and needs in areas such as the scientific basis and validation in some disciplines; the credentials and training of performers; funding and infrastructure; organizational independence; and the understanding, use and scrutiny by legal and judicial communities (NRC, 2009b). Dr. Murch expressed the hope that improvements in these areas will result in greater accuracy and defensibility of the science of forensics, which will in turn support increased confidence in the results of forensic analyses.

Moving to the specific area of microbial forensics, Dr. Murch again emphasized the necessity of consistent analysis methods and for obtaining a sample of known origin for comparison with samples collected as evidence in an investigation. Currently, the analyses used for microbial forensics are very context and situation dependent. There are a large number of pathogens that can negatively affect human, animal, and plant health, and for the vast majority, the appropriate method for forensic analysis has not been worked out or validated. Complicating matters further, there is a large array of genomic, physical, and molecular analytical methods that are available for use, but in some cases, the limitations of those analyses for characterization and identification purposes are not yet clear. In addition, Dr. Murch noted that he expects advances in analytical methodology to continue to occur at a rapid pace, and to provide new opportunities and new questions for forensic science.

In spite of these challenges, Dr. Murch noted that there is great potential for microbial forensics to be a critical tool to assist in the response to and investigation of terrorist attacks or hoaxes, support global non-proliferation efforts, aid in tracking and control of agricultural or public health outbreaks (either natural or of a suspicious origin), and other similar activities. In the future, microbial forensics could be used to help address the potential threats posed by engineered pathogens. However, to achieve its full potential will require the development of robust, effective, validated, and defensible analytical methodologies and the availability of samples of known origin. Since forensics is only effective when part of a broader legal and policy system, Dr. Murch suggested the development of a global, cooperative network in support of biosecurity and the development of a strategy within the BWC framework that could:

  • Identify “grand science challenges”
  • Establish standards/guidelines for collection, preservation, analysis and reporting and interpretation of results for organisms, toxins and sample types
  • Accept quality management standards
  • Provide standardized introductory and advanced training, personnel certification protocols for voluntary or selected participants
  • Establish one or more accredited UN-authorized microbial forensics laboratories based on international quality management standards, and modus operandi to include transparency
  • Establish accepted sample sharing, analysis protocols
  • Establish an international microbial forensics repository which leverages existing, related resources, to include specimens which help to describe relevant geotemporal microbial background
  • Develop and validate legal & policy requirements for microbial forensics capabilities that support UN--sponsored investigations, and then criteria and guidelines for use of science in UN actions

Trends in Biosensors – Gary Resnick, Los Alamos National Laboratory, USA

Dr. Gary Resnick began his talk by providing a general overview of biosensors as an analytical device designed for detection, and sometimes identification, of a biological analyte. Biosensors contain three key components: a biologically sensitive element, a transducer, and a signal processor. There are many available variations of these elements, some based on recognition (binders, protein engineering, etc.) and some based on transduction (electrochemical, optical, etc.) (Luong et al., 2008). These detectors are found in many areas including public health, agriculture, food safety, and security. Today, most biosensors can be found in central laboratories. In the future, Dr. Resnick hypothesized that detectors will become portable and more available for on-site, field-based analysis.

Encouraging the development of new biosensors, Dr. Resnick described the pull of societal needs, such as climate change and increased food production, and the push of new technology opportunities provided by the convergence of diverse technological developments in engineering and biological sciences and the drive for high-throughput bioanalysis systems. Dr. Resnick speculated that these developments are likely to see a growth and improvement of biosensors and, especially when coupled with advances in the area of systems biology, a veritable tsunami of biological information that will be a significant challenge to manage and process. In particular, he highlighted two advances that would be transformational for the field: the development of handheld biosensors and the development of “dipstick” technology—very simple-to-use, inexpensive sensors.

Advances in materials science, bioengineering (synthetic ligands, etc.), high-throughput genetic screening methods, and other fields will likely result in the development of new, faster assays, novel sensor platforms, and more efficient use of materials and reagents, perhaps even with the development of more robust, hand-held, self-contained detectors. However, no single biosensor will be able to address the requirements for all fields, and the development of biosensors in the future will require consideration of the need it is designed to meet. For example, a system designed for biodefense purposes will likely put greater emphasis on portability and high sample throughput than a system designed for diagnostic purposes for the public health arena. Dr. Resnick observed that creating an integrated biosurveillance system that effectively incorporates biosensor-acquired data will also require the development of an iterative process that uses available data streams, information and knowledge management, and knowledge of applications to identify and develop technology to meet to changing needs. Finally, Dr. Resnick emphasized that biosensors may be designed to detect known threats, even a broad range of known threats, but it should be understood that this does not provide absolute detection capability, and new threats or unexpected technological developments should not be a surprise. Biosensors are only part of a larger response and information infrastructure to protect against biological threats, whether naturally or intentionally introduced.

Biosensor Development – Ilya Kurochkin, M.V. Lomonosov Moscow State University, Russia

Dr. Ilya Kurochkin, from Lomonosov Moscow State University (MSU), provided participants with an overview of biosensor development efforts at his institution. The research is in the following areas: scanning probe microscopy for detection and identification of toxins and microbiological objects; development of an electrochemical bioanalytical platform; development of silicon nanowire transistors; and creation of a lateral flow device with surface-enhanced Raman spectroscopy (SERS) and enzymatic metallography amplification.

First, Dr. Kurochkin described the development of an antigen-antibody binding technique for improved scanning probe microscopy of proteins. In this method, a polymer layer containing antigens for a given biological species is created on a surface. Incubation of this material with the analyte allows for selective aggregation of proteins on the surface of the polymer. These can then be counted directly using scanning probe microscopy. He reported improved analytical sensitivity with this technique than with other methods and applicability to large proteins (over 8 nanometers).

Second, Dr. Kurochkin discussed the development of two analytical methods that lend themselves to use in array-based detectors: electrode-based sensors and silicon nanowire transistors. The electrode-based sensor array can be used for monitoring of neurotoxins and biological species. In this method, manganese dioxide (MnO2) nanoparticles are placed on the tip of an electrode and run through a series of alternating solutions and washes to create ionized monolayers on the surface. These monolayers can be created to be sensitive for specific compounds of interest and, when exposed, their reaction will cause changes in the current passing through the electrode. These changes can be detected and amplified when incorporated into a detector system. To provide multiplex capability, electrodes can be primed with different solutions and placed in an array. Dr. Kurochkin noted that these systems are currently in use for monitoring and testing in blood analysis and for detection of chemical weapons. He also noted the potential benefit that self-assembly methods have for creating large numbers of low-cost, reproducible sensors for a variety of applications. Next, Dr. Kurochkin described the development of silicon nanowire transistors for high sensitivity detection of ions in solution. By carefully placing a solution droplet onto the transistor one can monitor the affinity of the ions in solution to the transistor by changes in the current through the transistor. As a result, these can be used as detectors for solutions with very low concentrations of analyte.

Finally, Dr. Kurochkin described some advances in the area of surface-enhanced Raman spectroscopy (SERS), a non-destructive, optical technique often used for the study of biological systems. This is a powerful technique, but has low sensitivity, in part due to high background signals from samples. One method for increasing sensitivity is to put the sample on a metal surface, which enhances the Raman signal. It is not uncommon today to use metal nanoparticles to provide the enhancement. At MSU, improved methods for creating thin films of nanoparticles have been developed to improve SERS signals. In addition, improved sensitivity has been demonstrated by combining nanoparticle-based SERS analysis with a lateral flow device.

Remarks: Science Used in Identifying the Anthrax Mailings – Nancy Connell, University of Medicine and Dentistry of New Jersey, USA

Dr. Nancy Connell, from the University of Medicine and Dentistry of New Jersey, presented an overview of the 2001 anthrax attacks and the subsequent FBI investigation. She noted that during September and October, 2001, seven letters were mailed to media and senate offices in New York, Florida, and Washington DC, leading to contamination of 35 postal facilities and mailrooms, 22 cases of anthrax infection resulting in 5 deaths, and the treatment of 10,000 people judged to be potentially “at risk” with medical prophylactics. The subsequent investigation included the cooperation of 29 laboratories and the collection of 5,730 environmental samples taken from 60 sites. Dr. Connell highlighted some of the scientific techniques used during the investigation, including forms of microscopy to identify physical characteristics of the mailed spores, and forms of spectroscopy to identify chemical composition of the materials. In particular, she noted that the anthrax strain used in the mailings was determined to be the “Ames” strain and that genetic analysis was conducted to identify rare mutations in the mailed spores. Genetic screening was then undertaken to compare these mutations with the genetic sequences of reference anthrax samples from 15 domestic and 3 overseas laboratories. The presentation provided context for an ongoing National Research Council study that was examining the science behind the techniques used to identify the origin of the anthrax spores. Dr. Connell presented the statement of task for the committee and summarized the findings of the public FBI report on the investigation. As the NRC committee’s work was ongoing at the time of the workshop, Dr. Connell was unable to present any findings from that study.15

Discussion

The discussion that followed highlighted some of the challenges of using detectors alone to monitor for disease outbreak or potential attack. These include the size of detectors; the cost of broad sampling, whether environmental or in a public health setting; the cost and time required to process many samples; observing an emerging signal above the noisy microbial background; lack of availability of known samples for comparison purposes; and the difficult balance between creating a system designed to monitor for a specific threat versus one designed to monitor for a broad range of microbes. Many participants expressed the general sense that significant progress has been made in this area in recent years, and the development of hand held detectors and reduced sequencing costs could result in greater availability of microbial detectors and a better understanding of the background microbial world in the next decade.

Footnotes

15

The report, Review of the Scientific Approaches Used During the FBI's Investigation of the Anthrax Letters (NRC 2011b), has subsequently been published and is available at http://www​.nap.edu/catalog​.php?record_id=13098.