Optical Sensing Technology for Environmental Immunoassays - ACS

Chapter 10

Optical Sensing Technology for Environmental Immunoassays

Downloaded by NORTH CAROLINA STATE UNIV on October 11, 2012 | http://pubs.acs.org Publication Date: October 23, 1996 | doi: 10.1021/bk-1996-0646.ch010

Stephen L. Coulter and Stanley M. Klainer FCI Environmental, Inc., 1181 Grier Drive, Building B, Las Vegas, NV 89119

Immunoassay kits have been developed for field use in environmental monitoring. While they satisfy most of the requirements for an effective field method, they still suffer from the complexity of their use, and the difficulty in obtaining a reproducible endpoint. The marriage of competitive immunoassay to optical sensors results in a simple, solid-state system which provides a single step method for environmental monitoring.

Needs for environmental monitoring are constantly increasing. Effective environmental monitoring requires the use of field analytical instrumentation. Environmental monitoring is typically a transitioning of laboratory analyses to field analyses. Most analytical methods used today are not suitable for the field. They were primarily designed for laboratory operation and lack the critical elements necessary for field environmental analyzers: 1) Superior performance characteristics; 2) Operational methods which niaximize the performance of the analyzer (ease of use); 3) In situ measurements (no reliance upon sampling and preparation); 4) Real-time responses; 5) No sampling artifact; 6) Low cost for evaluation of multiple samples (encourages broader testing schemes); 7) Portability. While some of the existing monitoring equipment has characteristics which make them useful for field use, a major barrier is still the ability to perform analyses in situ. Fiber optic sensors using either chemical or biological sensing mechanisms are inherently wellsuited for in situ methods. The development of solid-state, fiber optic instrumentation based upon fluoroimmunoassay techniques provides not only a higher level of performance characteristics (sensitivity, selectivity) over other environmental, field instrumentation, but also provides a field methodology which is extremely simple to use.

0097-6156/96/0646-0103$15.00/0 © 1996 American Chemical Society In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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ENVIRONMENTAL IMMUNOCHEMICAL METHODS


FOCS® SENSORS FCI Environmental has designed products based upon a fiber optic chemical sensor (FOCS®) technology. The FOCS® sensors utilize optical waveguides (e.g., fiber optics) coated with proprietary sensing chemistries which are designed to interact with specific environmental contaminants. The sensing package comprises a light source which provides the output through the waveguide, the sensing waveguide and the appropriate detector. The light transmission through the waveguide is based upon the phenomenon of total internal reflectance. The transmitted light interacts with the sensing chemistries on the surface of the waveguide. This interaction directly affects the signal transmitted to the photodetectors. The FOCS® design can accommodate a number of optical sensing mechanisms and a number of detection methods. The sensing mechanism can be based upon organic, inorganic or biologic responses. The method of detection can be based upon any of the common optical detection methods including those listed below.

SENSING MECHANISMS Organics

Antibodies/Antigens

Dyes

Enzymes

Polymers

Biologies

Inorganics

Organometallics

II DETECTION4 METHODS Fluorescence

Refraction

Absorbance

Chemiluminescence

The current PetroSense® instruments are based upon the FOCS® technology. Detection is based upon refractive index changes as hydrocarbons interact with the fiber optic. The PetroSense® sensors detect total petroleum hydrocarbons (TPH) in the parts per million range. They meet the requisite performance criteria for field analyzers. They have the necessary sensitivity and selectivity for field screening instruments. The primary advantage that the PetroSense® analyzers have is their mode of operation. The PetroSense® sensors operate in vapor, in water and at the interface between vapor and water. They provide real-time, in situ, analytical measurements of environmental contaminants at low cost. By taking in situ measurements, the need for sampling, storage and transportation is eliminated. The greatest source of error in typical field monitoring methods comes form this sampling and handling (1). The ability to have the data in real time enables more accurate mapping of a contaminated site by allowing the field investigator to know when additional data is needed at the time of

In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

10. COULTER & KLAINER

Optical Sensing Technology & Immunoassays

evaluation. If these additional analyses are delayed and data/samples are collected at different times, artifact is introduced into the comparisons due to the temporal changes in the contamination site. The combination of low cost per analysis, ease of use and realtime results encourages a more thorough site analysis.


COMPETITIVE IMMUNOASSAY OPTICAL SENSOR Whereas the current PetroSense® sensors meet all the criteria for effective field screening instruments, the goal of FCI Environmental is to improve both the selectivity and the sensitivity of these sensors in order to provide analytical sensors. The current sensors detect a composite T P H concentration in the parts per million range (0.1 to 40,000 ppm of TPH.) The goal is to be able to detect parts per billion levels of selected contaminants and to be able to selectively detect specific contaminants or classes of contaminants. Immunoassay methodologies provide the selectivity desired. By utilizing a fluorescent detection scheme, the sensitivity of the sensor is improved to less than a part per billion. Immunoassay kits have been commercialized to take advantage of the specificity of the method. Table I provides some examples (not an exhaustive compilation) of commercial kits used for field screening: Table L Commercial Environmental Immunoassay Kits Target Analyte

Companies Involved in Environmental Immunoassavs

BTEX

D-TECH EnSys Millipore

Quantix SDI

PAHs

D-TECH EnSys

Millipore Quantix

Chlorophenols

EnSys Millipore

Ohmicron

PCBs

D-TECH EnSys

Millipore Ohmicron

TNT

D-TECH EnSys

Millipore Quantix

Dioxins

EnSys

SDI

Pesticides

Millipore Ohmicron

Quantix

The problem is that most field screening kits require multiple steps (typically 6 to 10 steps), have significant time delays (often 20 minutes) and are difficult to use in terms


105


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DISPLACEMENT

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Figure 1. FCI Environmental Competitive Immunoassay Scheme

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e.oH

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Figure 2. Competitive Immunoassay Response

Figure 3. Chip Level Waveguide Sensor



10. COULTER & KLAINER

Optical Sensing Technology & Immunoassays

107

of obtaining an accurate, reproducible endpoint. The strategy of FCI Environmental is to develop a solid-state, single step immunoassay sensor for environmental analyses which requires less than 5 minutes per assay. The selectivity of the immunoassay, the sensitivity of fluorescence detection and the ease of use with a single step, solid- state configuration may provide the next major breakthrough for environmental field screening instrumentation. The immunoassay techniques employed at FCI Environmental are based upon competitive immunoassay methods. Some of the considerations in the development of a competitive immunoassay sensor include the lack of sensitivity due to high background noise, inadequate antibody loading on the substrate and inefficient exchange of the environmental analytes (antigens) for the "tagged" analytes on the sensor. In a competitive immunoassay optical sensor, the desired antibody is immobilized to the optical waveguide. Tagged antigens or analytes are bound to the antibody, with a "tag" typically being a fluorophore. When the environmental analyte comes into contact with the sensor, it displaces the tagged anaryte, causing a change in the fluorescent signal intensity. The company's first demonstration of the technique was for a "drugs of abuse" application (2). The antibody for cocaine was immobilized to the sensor surface, followed by the complexation of the antibody with fluorescently tagged cocaine. When the sensor was exposed to cocaine in a sample (as in Figure 1), the competitive reaction resulted in a loss of the fluorescent signal. The detection limits of this sensor were determined to be less than 400 parts per trillion (see Figure 2.) The fabrication of the solid-state immunoassay optical sensor utilizes monoclonal, polyclonal or pooled monoclonal antibodies to establish the selectivity necessary for a given application. The selection of the tag for the anaryte controls the efficiency of exchange between the analyte in the sample and the initially bound analyte on the sensor. Standard coupling methods were applied to the system to generate membrane-based sensors and FOCS® sensors. The efficiency of these coupling methods was maximized for the highest analyte loading.

"IMMUNOASSAY ON A CHIP" To maximize the efficiency of the solid- state immunoassay sensor, a chip level waveguide sensor platform has been developed (3). This chip sensor incorporates the light source, optical waveguide and photodetectors into a single sensing element. The chip is designed to be "dual-armed" with a sensing arm and a reference arm It can also be fabricated with a single arm, or even multiple arms for a variety of sensors on a single platform. There are several advantages of the chip design in the areas of size, performance, manufacturabihty and cost. The smaller design allows for the reduction in the size of the overall probe. The performance of the sensor is improved due to an increase in sensitivity and a reduction of artifact (noise). Signal intensity has been improved by reducing the number of interfaces in the design and through the configuration of the sensing element which minimizes the amount of lost light. The incorporation of a referencing channel with balancing of the incident fight signal which allows for the subtraction of any humidity and temperature effects, minimizes the level



108


of artifacts (noise). Additional noise can come from undesired association of the tagged analyte with the sensor. Efforts were made to increase the yield in the binding of the tagged analyte and then to block the remaining sites with bovine serum albumin. This served to minimize the unused active binding sites, a significant source of background noise. The design characteristics of the optical chip configuration are designed to address the level of background noise which is the other barrier to improved performance mentioned earlier. The manufacturability of the chip design is a major benefit. The complexity of the assembly process is reduced based upon the incorporation of several components into the single chip. Issues involved with reproducibility of the alignment of the components are greatly reduced with automated fabrication of the chips. The yield of the process is increased, the inspection of the goods is simplified and the overall efficiency is improved The obvious result of these improvements in sensor performance and manufàcturability is a cost reduction for the sensor. The current focus at FCI Environmental is the adaptation of the FOCS® immunoassay configuration to the chip. Two products have currently been developed on the chip for petroleum hydrocarbons and for indoor air quality monitoring. The use of immunoassays will allow the development of a large number of environmental sensors based upon simply changing the antibodies on the chip. With a common platform, the sensors become interchangeable for applications to areas such as indoor air quality, industrial hygiene or personal exposure monitors.

Literature Cited 1.

2.

3.

Field Measurements, Dependable Data When You Need It; United States Environmental Protection Agency, Solid Waste and Emergency Response; EPA/530/UST-90-003: September, 1990. L i , H . ; Goswami, K.; Klainer, S. The Use of Fiber Optic Chemical Sensors (FOCS) For the Detection of the Effluvia from Illicit Drugs and the Materials Used to Prepare These; Final Reportfor Phase I; Advanced Research Projects Agency Contract No. D A A D05-92-C-0022: December, 1993. Saini, D.P. Chip Level Waveguide Sensor; U . S. Patent No. 5,439,647; August 8, 1995.


Optical Sensing Technology for Environmental Immunoassays - ACS

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