Sensor Systems for Biological Agent Attacks Protecting Buildings and Military Bases (2005) / Chapter Skim
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7 Structure-Based Identification for Detect-to-Warn Applications
7 Structure-Based Identification for Detect-to-Warn Applications
Pages 105-129
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From page 105... ...
To achieve this goal, one should use high-afflnity molecular recognition elements, reduce nonspecific binding by appropriate selection of the material that comes in contact with the sensor system, and employ high-sensitivity detection methods. Table 7.1 lists some transduction methods used in biosensors.
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From page 106... ...
In these signal transduction systems, the cell produces and displays its molecular recognition elements on its surface, embedded in its membrane. Each such element binds a specific tanget, usually to an extent that neglects the amount that is present.
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From page 107... ...
In addition to antibodies, a wide variety of other molecular recognition elements can be used for biosensing,4 some of which are summarized in Table 7.2. Many of these molecular recognition elements are proteins (e.g., enzymes, ledtins, receptors)
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From page 108... ...
Thus even if the cell surface were disnupted, the surface stnuctures would remain intact, albeit on many particles (rather than one) , and would retain their ability to bind the molecular recognition elements.
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From page 109... ...
If there are multiple binding steps required for detection, the time constraints for each binding step are even more severe. There have been many reports investigating the kinetics of binding targets to molecular recognition elements immobilized onto biosensor surfaces.5 These results show that under flowing conditions and with sulk cient target concentration, detectable one-step binding can be achieved in a few seconds.575 However, the results to date indicate that 1-minute analysis time is a challenge and will require a sensor design that minimizes the number of binding and processing steps and enhances mass transport of the target to the sensor surface.
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From page 110... ...
Research to detemmine the appropriate biomarkers for bioagents of interest and develop stnuctural recognition elements for those biomarkers is critical for selective bioagent detection. Another important consideration for sensor specificity is the binding of untargeted substances to the sensor surfaces.
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From page 111... ...
This detection limit has been demonstrated using several stnucturebased detection systems currently available. Sample Collection · Collect the sample for 400 seconds from the air into an aqueous solution using a two-stage, precollection fractionator with an overall capture efficiency of 50 percent and a collection rate of 90 liters of air per minute into 50 microliters of aqueous volume ° An air sample with 100 target structures per liter would be concentrated to 30,000 spores in a 50 microliter solution.b - Bind molecules to the sensor surface over 20 seconds via an active transport mechanism (e.g., pressure, electrophoretic transport, ultrasonic focusing)
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From page 112... ...
, the false alarm rate due to this factor would not be improved. (However, a control sensor surface without the selective chemistry can be used to normalize the sensor signal and minimize the effects of matrix materials.)
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From page 113... ...
The use of reporter groups adds some complexity to the system, because separate steps are typically required for their binding and also for washing to remove nonspecifically bound and unbound reporters from the sensor surface. Any additional binding steps are governed by the same transport and reaction kinetics described above for the binding of the molecular recognition element.
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From page 114... ...
The stringent washing must remove bound target and nonspecifically bound materials but leave the molecular recognition element unaffected. Renewal of the sensor surface is typically only possible for a limited number of cycles, after which time the sensor surface must be replaced due to degradabon.'5 Therefore, molecular recognition elements that can withstand harsh washing procedures are desirable to enable repeated renewal of the sensor surface.
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From page 115... ...
These estimate the analysis time for two different detection limits: (1) one femtomolar (about 10,000 targets bound to the sensor surface, see Box 7.1 )
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From page 116... ...
. Antibodies The classical molecular recognition elements, antibodies, are produced by most vertebrates.
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From page 117... ...
A variety of technical modifications such as the cross-linking of two aptamers that bind to different parts of a target are being investigated 2~ Aptamer technology provides additional opportunities in terms of the design of molecular recognition elements that could have affinities and selectivities that complement those of the more traditional antibody reagents. In some cases DNA aptamers have been reported to have higher affinities than antibodies.25 Aptamers also are reported to have greater stability and a longer shelf life than proteins Peter Biggins, Dstl.
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From page 118... ...
As with antigen-anbbody systems, the rate of detection depends upon the roncentrabon of the target, mass transfer of the target to the sensor surface, and the number of processing steps required for detection. Aptamer binding to targets, while nommally noncovalent (as is anbbody-target binding)
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From page 119... ...
The peptide has been shown to bind to the SpsC protein of the spore.32 Similar library constnucton and selection techniques could be employed to identify short peptide molecular recognition elements for other targets or other surface features of B anflhracis.
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From page 120... ...
Even if the disposables are minimized and these systems are reconfigured into an automated fommat for aerosol monitoring, the detection limit for these systems is currently too high for detect-to-wam applications. At least 10,000 targets bound to the sensor surface are required for detection, and the analysis time is 15 minutes or longer.
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From page 121... ...
onto a 1 square millimeter sensor surface, this detection limit corresponds to the binding of more than 1 Or targets onto the sensor surface. This detection limit is about two orders magnitude higher than immunoassay s Persson, K
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From page 122... ...
Flow Cytometry Cell biologists have for many years used flow cytometers—devices that identify, count, and sort cells on the basis of preselected properties 4~ in a sensor application, target cells are provided with fluorescently labeled molecular recognition elements (e.g., antibodies) , and a stream of individual cells is passed through the detector for analysis 47 Since flow cytometry analyzes single cells, the detection of 300 cells per 50 microliters, as described in the notional example in Box 7.2, is routinely achieved using this approach.
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From page 123... ...
Methods are also under development for enabling unattended operation of bead-based assays using flow cytometers.59 Bead suspension arrays were recently demonstrated for the simultaneous detection of four different bioagent simulants.5' The detection limits obtained with a total analysis time of about 1 hour (starting with a liquid sample) were about 5 x 104 ctu per milliliter for Erwinia herbicola, 1.5 x 104 ctu per milliliter for Bacillus globigii, 4.2 x 107 cfu per milliliter for MS2 (an RNA bacteriophage that is a simulant for smallpox vinus)
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From page 124... ...
to develop approaches to improve detection limits, especially for toxins and viruses. Target Binding That Changes Detectable Properties of Smart Sensor Surfaces Another general type of sensor system that is attractive for rapid detection, as required for detect-towam applications, is a sensor that is designed so that its surface changes in a detectable way only upon specific binding of the target to the sensor surface.
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From page 125... ...
The sensing surface is engineered so that the confommational change in the aptamer results in a change in fluorescence intensity and/or fluorescence spectra of the sensor surface.53 in one model biosensor system tested for thrombin detection, the detection limit was 5 nanomoles (0.7 attomole flhrombin in 140 picoliters) and flhe analysis time was 10 minutes.54 Given that single molecule fluorescence detection has been demonstrated in benchtop fluorescence detection systems with properly engineered smart sensor surfaces, it is conceivable that approaches based upon fluorescence detection will be able to detect 100 bound molecules (notional example in Box 7.2)
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From page 126... ...
Given the low detection limit and rapid response of this approach, detection of 10 to 100 ACPLAs should be achievable in seconds (see notional example in Box 7.2) , and a total analysis time within 2 minutes for detect-to-wam applications should be possible.
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From page 127... ...
Although an order of magnitude improvement in the detection limit for cells would be desirable for detect-to-warn applications, the 1 x 104 to 10 x 104 cfu per milliliter detection limits currently achieved using this approach may be suitable if aerosol collection times are approximately 1 minute (rather than 4 seconds, as listed in the notional example in Box 7.2) and the liquid volumes are scaled up to handle the quantities required for the assay.
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From page 128... ...
The challenges for rapid detection for such detect-to-wam applications include accelerating the transport of the target to the molecular recognition element, decreasing flhe number of processing steps to speed detection, and improving detection sensitivity (detection of as few as 100 targets bound to the sensor surface is needed)
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From page 129... ...
It should be based on performance requirements such as affinity, specificity, speed, false positive rate, cost, manufacturability, and other criteria.
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