Battling bio-terrorism

In the wake of the terrorist attacks last year there is a sudden demand for effective diagnostic tools for the rapid identification of biowarfare agents, not only for the military sector but for the civilian sector. Bioterrorism presents some difficult technical challenges for detection. Not only is there a wide range of bioagents to identify, but the demands of field use place constraints on the complexity and size of the sensor devices and attendant instrumentation. For these reasons, a "one size fits all" answer to the problem is unlikely, and there is a need to look outside the box of existing technologies for some of these answers.

The ideal biosensor for counter-terrorism and defense applications needs to have high sensitivity and wide applicability to different types of biowarfare agents, while remaining compact, easy to use, fast, and reliable (no false positives). One type of sensor approach that shows considerable promise is optical refractive index sensing technology, based, for example, on interferometry or surface plasmon resonance. These devices function by measuring the effect on optical waves of a coherent light source produced by the refractive index changes associated with biomolecular binding at the sensor/solution interface.

Our engineers are developing an optical chip based on a proprietary interferometer design for the rapid detection of proteins, pathogens, and nucleic acids.^1-3 This optical biowarfare chip has been used for the trace level detection of a range of biomolecular analytes, including small proteins, large protein agglomerates, and whole microorganisms. Unlike other technologies, this technology also performs well for samples consisting of complex matrices, for example whole blood.

Figure 1. In interferometric optical chip (top view, left), boundtarget bioagents on the sensing surface interact with the evanescent wave from the waveguide layer to produce a phase change relative to a reference wave, indicating a bioagent. Gratings (side view, right) couple light on and off chip.

Inset: Light traveling down each sensing path (sample and reference) reflects off the total internal reflection (TIR) mirrors and onto the 50/50 Fresnel beamcombiners. The mixed light waves create two identical interference outputs that are processed off-chip.

The optical biowarfare chip uses plane waves to address multiple interferometric circuits fabricated into the chip using standard thin-film processes. Based on the Hartman interferometer, the chip is a planar structure that consists of a thin silicon nitride waveguiding film deposited on an optical glass substrate via plasma-enhanced chemical vapor deposition (see figure 1). A 40-nm silicon dioxide thin film deposited on top of the silicon nitride provides a surface for the subsequent chemical attachment of bioagent receptors such as antibodies or nucleic acid probes. These receptors bind to specific target bioagents such as viral particles, toxins, or nucleic acid sequences. Grating couplers integrated into the supporting optical glass substrate couple light from a 670-nm diode laser in and out of the chip. Total internal reflection occurs at the dielectric interfaces such that light coupled into the waveguide layer is confined and guided.

This guided mode of coupled light possesses an evanescent field distribution that decays exponentially into both the substrate (bulk optical glass) and superstrate (biomolecular) layers. Through interaction with the evanescent wave, the binding of target bioagents to the receptors on the sensing surface produces a phase change in the guided light beam relative to a parallel reference light beam that passes through a region of the optical chip surface that is functionalized with a non-specific receptor. These two sensing regions form a single interferometer, or optical sensing circuit.

The degree to which binding affects the phase of the guided beams is dependent on the refractive index difference between the target bioagent and the sample solution. This signal is quantitatively proportional to the number of biomolecules bound at any time. Because chip fabrication involves standard photolithographic and thin-film processing technologies, we can fabricate multiple interferometric sensors on a single, 2 x 3 cm optical glass substrate. Current designs feature four or eight optical sensing circuits, and expansion to >=50 sensing circuits without increasing the size of the chip is easily feasible. The lack of polarization optics or moving parts simplifies the instrumentation requirements for these devices.

The interference pattern produced by mixing the specific and reference light waves is generated on-chip using the integrated optic total internal reflection (TIR) mirrors and beam combiners. Light traveling down each sensing path reflects off of the TIR mirrors and onto the Fresnel beamcombiners, which are designed to be 50% reflective and 50% transmissive. The wavefronts interfere to create two identical interference outputs.

To measure the phase change occurring at any moment in time during biomolecular binding to the optical chip, it is necessary to know the maximum and minimum of the interference signal. For this purpose, we use a lithium-niobate modulator (LiNbO^₃), which changes refractive index as a result of an applied voltage. A voltage applied to a comb structure of LiNbO^₃ placed between the laser and the optical chip can be used to step the interference signal from the measured value through the signal maximum/minimum, and thereby determine the phase at each moment in time.

Optical interferometers (and other refractive index sensors) have features that make them versatile platforms for biomolecular sensing. Because refractive index is a fundamental material property, the interferometer becomes a sensing platform capable of responding to almost any physical change, chemical species, or biological agent via a change to the selectivity of the surface layer. Because the sensor is measuring each binding event, this real-time detection is also inherently quantitative, which can be valuable in some diagnostic applications.

In addition, the penetration depth of the evanescent field is approximately half the wavelength of the coupled light wave, allowing discrimination between solution- and surface-bound biomolecules. The sensitivity provides perhaps the most exciting feature of these devices—their ability to provide real-time information about the presence of target bioagents without the prerequisite for either wash steps or secondary labels. In other words, the devices can perform easy, fast assays even in complex biological fluids such as whole blood. Such simplicity of operation is a key component for a real-world counter-terrorism biosensor in both armed forces and civilian use.

Biomolecules such as proteins and nucleic acids have a refractive index of between 1.45 and 1.48, compared to a refractive index of 1.33 for water. The refractive index sensitivity of an interferometer is 10^-6 to 10^-7 using only passive signal processing techniques, which allows these devices to measure a fraction of a monolayer of surface binding. This performance configuration yields a detection sensitivity of about 1 pg/mm².

Signal amplification methods can further increase sensitivity. In this approach, a high-refractive-index nanoparticle, preferably gold, is conjugated to a receptor (antibody or nucleic acid probe) different from the capture receptor immobilized on the chip. These gold nanoparticle conjugates can be used in a number of different assay formats, such as a sandwich immunoassay. Only the antigen-gold conjugate complex that binds to the receptors on the optical chip is detected, and not the excess gold conjugate in solution, so unlike most sensors, the simple mix-and-read format is still possible. In addition, because the signal for the gold particle is two to three orders of magnitude greater than that for most proteins or short nucleic acid sequences, the size of the biomolecule becomes irrelevant. Active signal processing techniques can further improve performance.

Figure 2. The image shows results for a rapid immunoassay in human whole blood using human chorionic gonadotropin (hCG) as the target antigen.

Many of the technologies under development for counter-terrorism focus on nucleic acid (DNA) detection. However, a number of likely biowarfare agents, such as botulinum toxin, ricin, and Staphylococcus enterotoxins are proteins. Optical interferometry can be used equally well for the detection of any biomolecule, making it a versatile platform for both protein and DNA detection, with the possibility of performing both types of assays with the same basic instrument (see figure 2).

The real dangers posed by bioterrorism to both civilian population and military personnel require national efforts in the development of new counter-terrorism measures, including rapid diagnostic technologies. Optical biowarfare chip technology appears to possess all the necessary characteristics required to play a part in this major effort. oe

References

1. Hartman, N.F. US Patent 5,633,561 1997.

2. B. Schneider, E. Dickinson, et al, Biosensors Bioelectron. 2000, 15, 597.

3. B. Schneider, F. Quinn, ACS Symposium Series No. 815, Microfabricated Sensors: Applications of Optical Technology for DNA Analysis, Eds. Kordal R, Usmani A, Law WT, American Chemical Society, Washington, DC. 2002.