Nanoscience and Nanotechnology for Chemical and Biological Defense

Chapter 6

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Hand Held Biowarfare Assays Rapid Biowarfare Detection Using the Combined Attributes of Microfluidic in vitro Selections and Immunochromatographic Assays Letha J. Sooter1, Dimitra N. Stratis-Cullum1*, Yanting Zhang2, Jeffrey J. Rice2, John T. Ballew3, Hyongsok T. Soh3, Patrick S. Daugherty3, Paul Pellegrino1, Nancy Stagliano2 1

US Army Research Laboratory, AMSRD-ARL-SE-EO, 2800 Powder Mill Road, Adelphi, MD 20783 2 CytomX, LLC., 460 Ward Drive Suite E-1, Santa Barbara, CA 93111 3 University of California, Santa Barbara, Santa Barbara, CA 93106

Immunochromatography is a rapid, reliable, and cost effective method of detecting biowarfare agents. Gold nanoparticles provide a visual indication of target presence. A fundamental limitation for this type of assay is the availability of molecular recognition elements (MRE’s) which bind the target. Typical in vitro selection methods to produce the MRE’s require weeks or months of work. To alleviate this problem, microfluidic sorting chips have been developed to rapidly screen for binders.

Many people are familiar with immunochromatography (1-3). Over-thecounter pregnancy tests are a prime example. These assays have a number of advantages. Two of their most important characteristics is that they are inexpensive to produce and disposable. They are also small, portable, and have © 2009 American Chemical Society

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In Nanoscience and Nanotechnology for Chemical and Biological Defense; Nagarajan, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.

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a long shelf life. These assays are easy to use because they can be read visually. One purple line indicates the assay is functioning properly and two purple lines indicate the target molecule is present. An example of this can be seen in Figures 1 and 2. The Army is interested in producing these immunochromatrography tests rapidly when new chemical or biowarfare threats are identified. In order to do this, in vitro selections must be performed in a timely manner and they must produce strong, selective binders for the target of interest. Typical selections take from weeks to months to be completed. However, a microfluidic sorting chip has been developed that rapidly screens libraries to identify binders.

Figure 1. Typical hand held assay test (4). (see page 1 of color inserts)

Introduction Immunochromatography A hydrophobic membrane is one of the primary components of an immunochromatographic hand held assay (4). As shown in Figure 2, two lines of white latex beads, the control line and the sample line, are immobilized through hydrophobic interactions on one end of the membrane strip. The control line is composed of latex beads conjugated to one member of a two molecule binding pair, such as an immunoglobulin (IgG) and ProteinA. The latex beads in the sample line are similar in that they are conjugated to a binding molecule, but this molecule binds one of multiple epitopes on a target. A target


75 may be a small chemical, a large protein, or something as complex as cells or spores. The binding molecules are typically antibodies, but they may also be peptides or nucleic acids.

A.


B.

C.

D.

Figure 2. Schematic of positive and negative hand held assay tests. A. Target is present in the applied sample. B. The presence of target results in the formation of two purple lines on the test. C. No target is present in the applied sample. D. The lack of target results in the formation of one purple line on the test (4). (see page 1 of color inserts) On the opposite end of the membrane is a mixture of gold nanoparticles. Gold nanoparticles are a purple color and provide a visible signal when immobilized on the line of latex beads. The nanoparticles are composed of two populations. One complements the two member binding pair of the control line. The other binds a second epitope on a target. Therefore, regardless of whether a target species is present, the control line will turn purple upon application of a liquid to the membrane. When a target is present, it flows through the gold nanoparticles conjugated to specific binders and becomes attached. The nanoparticle/target pair then travels through the membrane via capillary action. The sample line will turn purple when the conjugated latex bead binds a second epitope on the target. In the absence of target in the sample, the gold particles will flow past the latex beads and no signal will be visible. In Vitro Selections In vitro selections are a powerful means of producing ligands with high affinity and specificity for a target of choice. The molecules which bind the


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target of interest in an immunochromatographic hand held assay are typically isolated through in vitro selection. These selections may utilize, for example, DNA, RNA, peptides, or antibodies, each with their own attributes. E. coli bacterial libraries displaying peptides have a particularly short time period required for library generation/regeneration, see Figure 3. As an example of an E. coli surface display peptide selection, the process begins with a library. Each bacteria displays a different peptide to create a population, or library, of approximately 1010 unique cells (2). This library is incubated with a labeled target for a period of time to allow binding between the peptide and the target. Following this incubation, the assay is typically loaded into a Fluorescence Activated Cell Sorter (FACS). This process separates the two populations which are present: E. coli and E. coli bound to target. The E. coli bound to the target is collected and placed in growth media allowing for amplification of the selected population for additional rounds of selection.

Figure 3. Advantage of bacterial protein display over other protein display methods (4). The instrumentation required for FACS is large and quite expensive. It may also take a significant time to sort samples. To alleviate these problems, a microscope slide sized, microfluidic sorter using a technique known as continuous trapping magnetic activated cell sorting (CTMACS) was developed as a new means of affinity reagent discovery, see Figure 4.



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Figure 4. CTMACS instrument. The combination of bacterial display libraries with CTMACS allows the rapid production of affinity reagents, see Figure 5. These, in turn, are used in the production of immunochromatographic hand held assays. The result is high throughput production and deployment of simple to use assays for the most relevant biological and chemical warfare agents.

Experimental Methods Immunochromatography Immunochromatographic hand held assays are conceptually straightforward, but require significant optimization (4). Whatman FUSION5 membranes were chosen because of their “one membrane, five functions” technology. These membranes eliminate the need for multiple layers of different membranes to construct a laminar flow, immunochromatographic, hand held assay. They are highly absorbent and require little or no blocking. A BioDot platform equipped with a Biojet can be used to dispense the lines of latex beads. The result is clear, sharp signal lines. A mixture of the conjugated gold beads are deposited in a spot at the other end of the membrane. Then, the membrane is allowed to dry.


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Peptide affinity ligand

N


C Reagent

Figure 5. Bacterial display selection using a microfluidic sorting device (1). A target sample solution and a negative control are pipetted onto separate hand held assays near the gold nanoparticles. The sample with target should produce two purple lines and the negative control should produce one purple line. Any non-bound nanoparticles will continue to flow to the end of the membrane strip. In Vitro Selections with CTMACS Continuous trapping magnetic activated cell sorting devices are approximately the size of a microscope slide. In order to perform selections using these devices, the target of interest must be conjugated to a magnetic particle. The bacterial display library is then incubated with the conjugated targets to allow binding. Then the bacterial library and target solution is applied to the CTMACS device, see Figure 6. Buffer channels move the assay across a series of magnets, where magnetic particles with bacteria bound targets are captured at the edges between



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Figure 6. Diagram of a selection using a CTMACS device. (see page 2 of color inserts)

Figure 7. Magnetic particles trapped inside a CTMACS device. opposing magnetic poles, see Figure 7. Unbound bacterial cells will flow through the chip and into waste. The buffer composition and flow rate ensure that target binding occurs with high affinity and specificity. Following washing of the bound magnetic particles, they are released and collected. The captured bacteria are allowed to grow, from which the peptide binding sequences are obtained.


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Control mixtures were run through the CTMACS device to measure the enrichment of magnetic particles. In Figure 8, a solution which was 0.004% magnetic particles and 99.996% polystyrene beads was run through the CTMACS device. The recovered mixture was 97.26% magnetic particles.

Figure 8. Enrichment of magnetic particles using a CTMACS device.

Figure 9. Magnetic particle recovery based on flow rate in CTMACS device. Next, the recovery of magnetic particles was measured based on flow rate, see Figure 9. The recovery remained relatively constant despite an increase from 10mL/hr to 30mL/hr.


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Results


Positive Control

CRP Test

Direction of Flow

Latex Gold Latex Gold Protein A IgG C7 antibody peptide Protein A IgG C7 antibody C2 antibody Protein A IgG C7 antibody C6 antibody Protein A IgG C7 antibody C7 antibody Figure 10. Hand held assay test for C-Reactive Protein. The leftmost line is the positive control and the rightmost line is the sample line (4). Figure 10 shows the results of hand held assays constructed to detect Creactive protein (CRP). CRP was chosen as an initial target due to the degree to which it has been studied and the availability of multiple antibodies against it. Figure 11 demonstrates an alternative method of immobilizing a binding molecule. Instead of conjugating the binder to a latex bead, it is expressed on the surface of bacterial cells. The cells remain immobilized in the Whatman FUSION5 membrane where they are deposited.


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+ -

Gold nanoparticles

Sample addition

Before

During After


Direction of Flow

Figure 11. Cell based hand held assay for the detection of T7 antibodies. (see page 2 of color inserts) In this particular assay, cells expressing the T7 peptide on their surface are immobilized at the positive detection mark (green) and cells expressing a negative control peptide are immobilized at the negative mark (red). Streptavidin coated gold nanoparticles are placed and dried at the blue mark. An antibody solution containing the biotinylated T7-antibody is added at the gray mark and PBS wash buffer is added at the same location to allow for transport and migration of the nanoparticles and protein solution. The purple signal line indicates the presence of the target molecule.

Conclusions Each type of hand held assay has its advantages. When latex beads are used, the binding molecule must be isolated from the bacterial cell and conjugated to the bead. This is time consuming. However, the resultant stability of the assay makes it ideal for long term storage or exposure to harsher environmental conditions. The cell based hand held assays may not be as stable to harsh environments, but they can be developed much more rapidly. They are ideal for medical based diagnostics where they would be constructed and used in a controlled environment within a short time period. When coupled with the CTMACS technology, a powerful method of chemical and biological warfare detection is the result.


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References 1. 2. 3.


4.

Bessette, P. H.; Hu, X.; Soh, H. T.; Daugherty, P. S. Anal Chem. 2007, 79, 2174. Rice, J. J.; Schohn, A.; Bessette, P. H.; Boulware, K. T.; Daugherty, P. S. Protein Sci. 2006, 15, 825. Chiao, D. J.; Shyu, R. H.; Hu, C. S.; Chiang, H. Y.; Tang, S. S. J Chromatogr B Analyt Technol Biomed Life Sci. 2004, 809, 37. Sooter, L.J.; Stratis-Cullum, D.N.; Zhang, Y,; Daugherty, P.S.; Soh, H.T.; Pellegrino, P.; Stagliano, N. Smart Biomedical and Physiological Sensor Technology V, edited by Brian M. Cullum, D. Marshall Porterfield. Proceedings of SPIE Vol. 6759, 67590A. 2007.


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