Fixed polarizer ellipsometry for simple and sensitive detection of thin films generated by specific molecular interactions: applications in immunoassays and DNA sequence detection

¹ BioStar, Inc., Boulder, CO 80301.

² Department of Electrical and Computer Engineering, University of Colorado, Boulder, CO 80309-0425.
^a Address correspondence to this author at: BioStar, Inc., 6655 Lookout Rd., Boulder, CO 80301. Fax 303-530-6627; e-mail r_ostroff{at}biostar.com.

Abstract

Biological thin films may form on a surface by specific molecular interactions. The fixed polarizer ellipsometer (FPE) is a sensitive instrument that detects biological thin films either qualitatively or quantitatively. The design is simple and inexpensive. The assays are formatted on an optical surface, and the FPE detection is based on the phase shift of linearly polarized light after reflection through a thin film. We have constructed mathematical models of the FPE response to reflection through single-layer and two-layer films that agree closely with experimental data. Several biological assays have been measured with the FPE to demonstrate the application of this technology to clinical targets, including ultrasensitive immunoassays for hepatitis B surface antigen (0.1 ng/mL) and -fetoprotein (0.01 ng/mL) and DNA hybridization (0.5 fmol/µL target probe). A clinical study for detection of group A streptococcus from patient throat swabs demonstrated the qualitative application of the FPE to infectious disease targets. The flexibility and sensitivity of the FPE makes this technology suitable for numerous target analytes and applications.

Key Words: FPE, fixed polarizer ellipsometer • TMB, tetramethylbenzidene • DLC, diamond-like carbon • HBsAg, hepatitis B surface antigen.

Thin films may form on a surface as a result of molecular binding events, such as antibody-antigen interactions, receptor-ligand binding, or DNA hybridization. The fixed polarizer ellipsometer (FPE)³ is a simple, sensitive instrument for detecting thin films resulting from single-sample/single-analyte testing to multiple-sample/multiple-analyte arrays. The principle of the ellipsometric technique for measuring thin films has been in existence since the late 1800s (1) . The state of the art instruments today are microprocessor-controlled, rotating null ellipsometers. These ellipsometers can operate at multiple angles and wavelengths to determine film thickness and refractive index. These instruments accurately determine film thickness in the angstrom range. They are also quite complex, expensive, and delicate to maintain. In contrast, the FPE is a simple, inexpensive design with performance comparable with the null ellipsometer.

The principle of the FPE measurement is based on the phase delay of polarized light from reflection through a thin film. We have generated mathematical models to predict the response of the FPE for single-layer and two-layer reflections and compared these models to experimental data. We have also demonstrated the use of the FPE for a number of quantitative and qualitative biological applications, including immunoassays and DNA hybridization.

optical assay surface construction

The optical assay surface is composed of multiple layers. The base material can be any suitably reflective surface, such as a solid silicon wafer or a porous, plastic membrane coated with silicon. The base material does not need to be perfectly flat. As long as the surface roughness is less than the incident wavelength of light in thickness, scattering is not important. The next layer, the attachment layer, links the specific capture agent to the optical surface by covalent or noncovalent methods. For example, a siloxane polymer can be used to noncovalently attach capture antibody to a silicon wafer. The final layer of the optical surface contains the capture agent. Over 10 million antibody-coated surfaces have been produced successfully to date.

To begin the assay, a sample containing target analyte is applied. After a 2 to 10-min incubation, the surface is rinsed. The biological thin film is completed by application of an enzyme/precipitating substrate system, typically composed of analyte-specific binding molecules labeled with horseradish peroxidase, followed by a precipitating tetramethylbenzidene (TMB) substrate. Total assay times range from 5 to 30 min. The specificity of the reaction is controlled through the molecular interaction of the analyte with the capture and secondary binding reagents.

principle of fixed polarizer ellipsometry

The FPE is composed of simple, inexpensive components. The essential elements are a light source, two polarizers, and a detector (Fig. 1 ). The light source can be polychromatic with a band pass filter or a laser. Glass or plastic polarizers are acceptable. The detector may be a common silicon photodiode.

View larger version (15K): [in this window] [in a new window]   Figure 1. Diagram of a FPE showing the polarization state of light as it travels from the source, through the Polarizer, thin film, and Analyzer, to the detector. Arrows, polarization state of the light.

The principle of the FPE is diagrammed in Fig. 1 . The light source emits randomly polarized light, which is linearly polarized upon passing through the Polarizer. When the linearly polarized light interacts with the optical surface, there is a shift in phases of both the components parallel and perpendicular to the plane of incidence. The shift is usually not the same for both components; therefore, the emerging light is elliptically polarized. The amount of ellipticity induced depends on the optical properties of the base material as well as the thickness and refractive index of the overlying films (1)(2) . The angle of the second polarizer, also known as the Analyzer, is set to select a component of the elliptically polarized light such that the change in intensity of that component is proportional to the thickness change of the biological film. The intensity value, therefore, provides a measure of the quantity of target analyte bound to the surface.

modeling of the fpe response

The key components of the FPE can be set to customize the response to a specific assay platform and configuration. The flexible parameters include: light source and wavelength; angle of incident light; and Polarizer and Analyzer angles. An example of the effect of different settings for angle of incidence on a group A streptococcus immunoassay is shown in Fig. 2 . The slope of the response changes with small changes in the angle of incident light.

View larger version (14K): [in this window] [in a new window]   Figure 2. Effect of changing the angle of the incident light on the FPE response. The immunoassay is detecting dilutions of group A streptococcus antigen. , 50 °; , 56 °; , 60 °; , 70 °.

We have developed a mathematical model to predict the FPE response for single-layer reflections (3) . The model allows us to anticipate the effect of changing component settings and also provides a mechanism for choosing an instrument configuration for optimal response to a desired target analyte range. The intensity at the detector can be expressed as:

where I_in is the source intensity, r_tot,p and r_tot,s are the amplitude reflection coefficients parallel and perpendicular to the plane of incidence, respectively, and _p and _a are the respective Polarizer and Analyzer angles with respect to the polarization axis.

The magnitude of the period of the FPE response curve changes with a change of instrument parameters (angle of incidence and Polarizer and Analyzer angles) and the thickness and refractive index of the film. The measurement is most accurate when the change of intensity with change in film thickness is greatest.

We have tested the model by comparing the predicted response to experimental measurements for a series of thin-film calibrators with a refractive index of 1.41 and ranging from 23 to 1570 Å in thickness (3) . The calibrators consisted of a siloxane polymer film spun onto a silicon wafer and baked. The refractive index and thickness were determined by a Gaertner null ellipsometer (4) . The expected FPE response was modeled for two specific sets of instrument readings and compared with experimental measurements of the calibrators at those settings (Fig. 3 ). These two instrument settings were chosen to demonstrate opposite trends predicted by the model. An increasing intensity value is predicted with increased film thickness in one case and a decreasing intensity value with increased film thickness in the other. The latter condition may be desirable for competitive assays, where higher concentrations of target analyte would yield decreasing film thickness. The measurements closely fit the model, indicating that the FPE response can be tailored for a desired assay range. The slope of the response from 250 to 1000 Å predicts high sensitivity to small thickness changes in this range. Typical optical immunoassays generate 100 to 800 Å thickness changes for positive results, depending on analyte concentration and assay configuration. Thus, assays taking place on an optical surface with 200–500 Å of base materials are in the optimal range for sensitive FPE measurements.

View larger version (22K): [in this window] [in a new window]   Figure 3. Predicted model and experimental data for single-layer reflection. FPE settings for the Polarizer angle (p), Analyzer angle (a), and angle of incidence (1) are given.

We have also modeled two-layer reflections, which describe the behavior of the FPE on reflection through a film composed of two materials with different refractive indices. An example of such a film is a biological layer (refractive index, 1.41) formed on top of an optical coating, such as silicon nitride or diamond-like carbon (DLC) (refractive index, 2.0). The example in Fig. 4 compares the predicted FPE response to actual measurements for a layer of siloxane polymer (T-polymer) of varying thickness formed on a constant layer of DLC applied to a silicon wafer.

View larger version (27K): [in this window] [in a new window]   Figure 4. Predicted model and experimental data for two-layer reflection. FPE settings are given for angle of incidence, wavelength of light source, and Polarizer (P1) and Analyzer (P2) angles.

Interference occurs between light reflected from the upper and lower surfaces of the DLC layer. The resulting interference signal is due to the combination of light reflected from the upper surface of the biological layer and the light reflected from the lower surface of the DLC. This signal may be measured by the FPE and depends on the refractive indices and thickness of both the DLC and the biological layers as well as the refractive index of the silicon substrate. As in the one-layer model, the FPE response also depends on the settings of the Polarizer and Analyzer.

To study this two-layer thin film structure theoretically, we first calculated the total reflectance from the DLC layer, following the matrix approach described by Trotter et al. (3) . We then used this total reflectance to replace the surface reflectance from the bottom of the biological layer (used in the single-layer model) and calculated the total reflectance of the biological layer, which in fact is the total reflectance of the two-layer structure. The difference between the total reflectance of the DLC layer and the real surface reflectance at the bottom of the biological layer is that the former is a complex number that represents both the magnitude of the reflectance and the phase change, whereas the latter is a real number without phase change. The theoretical prediction matches experimental results closely (Fig. 4 ). These simulations also correctly predict that the instrument settings chosen in Fig. 4B (lower graph) are in the linear response range, whereas those in Fig. 4A (upper graph) are not. This modeling experiment confirms that the FPE response for multilayer surfaces can be predicted accurately. Therefore, assays using different attachment chemistries and optical surfaces can be modeled to optimize the FPE response.

biological applications

The FPE has been used to measure the results of prototype immunoassays and DNA hybridization reactions applied to optical surfaces. These assays demonstrate proof of concept for the utility of the FPE for clinical assays. The level of sensitivity for these prototype assays equals or surpasses published sensitivity for commercial automated immunoassay systems (5) . Typical CV values for FPE measurements range from 2% to 6%.

One illustrative example is a room-temperature, 30-min immunoassay for hepatitis B surface antigen (HBsAg). Dilutions of HBsAg in plasma were incubated on the antibody-coated surfaces for 15 min. After washing the surface, a monoclonal antibody to HBsAg, labeled with horseradish peroxidase, was incubated on the surface for 5 min. The surface was washed, and the precipitating substrate TMB was incubated on the surface for 10 min. The surface was washed, air dried, and measured with the FPE. This assay tested a range of HBsAg samples from 0.1 to 10 ng/mL. The 0.1 ng/mL specimen gave a signal that was 10-fold above that of the negative control (Fig. 5 ). The detection limit, calculated as 3 SD above the mean negative control reading, yielded a calculated sensitivity down to 4 pg/mL for this assay.

View larger version (9K): [in this window] [in a new window]   Figure 5. Immunoassay with FPE detection for HBsAg diluted in Plasma Diagnostic Base. The two sets of data represent duplicate experiments.

To investigate the application of the optical surfaces and FPE for nucleic acid detection, a simple DNA hybridization experiment was performed. A capture oligonucleotide was noncovalently bound to the optical membrane. The surface was then probed with the biotinylated complementary oligonucleotide, and the thin film was completed with an anti-biotin antibody-horseradish peroxidase conjugate and precipitating TMB. Total assay time was 30 min at room temperature. Concentrations of probe as low as 0.5 fmol/µL were detectable with the FPE.

Another immunoassay we have configured for the FPE is for -fetoprotein. This assay can be conducted by using two different protocols. One protocol is a 15-min assay, and the response range is from 0.01 to 1 ng/mL. Simply by shortening the assay to 6 min total, the measurable range shifts to 10–1000 ng/mL, which is more physiologically relevant for the clinical use of this liver cancer marker.

The FPE can also be applied to qualitative applications. The FPE measurements were collected from immunoassays performed to measure group A streptococcus from 107 patient throat swabs (Fig. 6 ). The cutoff discrimination between positive and negative samples was established as 3 SD above the mean negative value. The results were compared with the manual STREP A OIA^® test. The STREP A OIA test has been demonstrated to have performance superior to standard throat culture (6)(7)(8) . The FPE measurement results showed 98% correlation with the visual STREP A OIA test, indicating that FPE is suitable for qualitative infectious disease targets.

View larger version (14K): [in this window] [in a new window]   Figure 6. Patient throat swab testing for detection of group A streptococcus antigen by immunoassay. FPE detection plotted against sample number. Cutoff was established as 3 SD above the mean negative reading.

In conclusion, the FPE is a simple instrument capable of sensitive measurement of very thin films formed by specific molecular interactions at an optical surface. The potential applications for the FPE are numerous. The low cost and simplicity of the FPE makes it a suitable detection system for point-of-care applications. A simple system could be designed to measure a small number of analytes (2–10) from a single patient sample. The system would offer rapid, accurate results in a simple format. Another potential use for the FPE is interpretation of arrays of assay results. The arrays could be the product of multianalyte screening from a single patient sample or single- analyte screening from multiple samples. The assays may be infectious disease targets, allergen panels, genomic screens, or clinical chemistry analytes. FPE is also suitable for interpreting screening arrays designed to identify lead compounds for novel therapeutic applications.

Acknowledgments

We acknowledge the contributions of several people from BioStar for the instrument design and applications: David Becker, Bob Bilodeau, Lyndal Hesterberg, Phil McMahon, Ray Mondesire, and Arthur Ross.

References

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