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Optimizing a Waveguide-Based Sandwich Immunoassay for Tumor Biomarkers: Evaluating Fluorescent Labels and Functional Surfaces Harshini Mukundan,† Hongzhi Xie,† Aaron S. Anderson,† W. Kevin Grace,† John E. Shively,‡ and Basil I. Swanson*,† MS: J567, Physical Chemistry and Applied Spectroscopy, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, and Beckman Research Institute, City of Hope, 1450 East Duarte Road, Duarte, California 91010. Received July 8, 2008; Revised Manuscript Received November 25, 2008
The sensor team at the Los Alamos National Laboratory has developed a waveguide-based optical biosensor for the detection of biomarkers associated with disease. We have previously demonstrated the application of this technology to the sensitive detection of carcinoembryonic antigen in serum and nipple aspirate fluid from breast cancer patients. In this publication, we report improvements to this technology that will facilitate transition to a point-of-care diagnostic system and/or robust research tool. The first improvement involved replacing phospholipid bilayers used for waveguide functionalization with self-assembled monolayers. These thin films are stable, specific, and robust silane-based surfaces that reduce nonspecific binding and enhance the signal to background ratio. Second, we have explored four different fluorescent labeling paradigms to determine the optimal procedure for use in the assay. Labeling the detector antibody with an organic dye (AlexaFluor 647) in the hinge region allows for unusual signal enhancement with repeat excitation (at 635 nm) in our assay format, thereby facilitating a better signal resolution at lower concentrations of the antigen. We have also labeled the detector antibody with photostable quantum dots through either the amine groups of lysine (Fc, NH) or using a histidine tag in the hinge region of the antibody (Hinge, H). Both labeling strategies allow for acceptable signal resolution, but quantum dots show much greater resistance to photobleaching than organic dyes.
INTRODUCTION Breast cancer affects 200 000 women annually in the United States. The probability of survival is much higher with early diagnosis (95%) compared to metastatic disease (26%) (1). Current diagnostic techniques are, however, unsatisfactory when applied to early detection. For example, mammography can detect the tumor only when palpable, suffers from high false positive and negative rates, and requires confirmatory invasive biopsies as a consequence. In addition, it cannot be used on women less than 40 years of age because of intrinsically denser breasts in that population (2). Other methods are also either expensive (e.g., magnetic resonance imaging) or are associated with nonspecific results (standard immunoassays) (3). Thus, there is a need for a sensitive, specific, quantitative, rapid, and noninvasive diagnostic for breast cancer. Certain “biomarkers” are differentially expressed in cancer, typically very early in disease onset, and can potentially be used for detection. Since no single biomarker can effectively predict all stages of cancer, detection of a suite of these molecules is a requisite of any diagnostic strategy that utilizes them. However, current immunoassay techniques cannot accomplish this in complex, small-volume patient samples. Therefore, the American Society for Clinical Oncology does not currently recommend these markers for the early detection of breast cancer (4). The sensor team at the Los Alamos National Laboratory has developed a waveguide-based optical biosensor for the sensitive detection of such markers in a sandwich immunoassay format. * To whom all correspondence is addressed. MS J567, C-PCS, C-Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545,
[email protected], (505)-667-5814(phone), (505)-667-0440 (Fax). † Los Alamos National Laboratory. ‡ City of Hope.
The optical properties and assay procedures have been outlined elsewhere (5). Briefly, a sandwich immunoassay using a fluorescently labeled detector antibody is performed on the functionalized surface of single-mode planar optical waveguides. Only fluorescent molecules within the evanescent field of the waveguide (0-250 nm from the surface) are excited, facilitating optical separation of surface-bound entities from solution contaminants. This spatial filtering minimizes the background fluorescence associated with complex biological samples such as blood and sputum. We have previously demonstrated the application of this technology to the sensitive detection of breast cancer (unpublished data) (6), influenza viruses (7), and Bacillus anthracis protective antigen (5). We have successfully adapted the optical biosensor to the detection of carcinoembryonic antigen (CEA), a breast cancer biomarker in serum and nipple aspirate fluid from patients with abnormal mammograms (unpublished data) (6). CEA was detected in a small cohort of patient samples with excellent sensitivity (limit of detection ) 1 week), harsh environments such as elevated temperatures, or passage through an air-water interface. Furthermore, high concentrations of lipids or urea in some biological samples (such as urine) results in elevated NSB, which limits the successful application of PLBs to biodiagnostic approaches. To overcome these disadvantages, we utilized PEG-
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modified, silane-based thin films. These coatings facilitate covalent attachment and chemical flexibility, and minimize NSB. We have previously demonstrated the application of these surfaces to the sensitive detection of the Bacillus anthracis protective antigen. Alkane SAMs terminated with PEG groups have been used for minimizing NSB (8). Concurrent with these results, use of SAMs as the functional surface in our assay abrogates the necessity for blocking. As shown in Figure 2A,B, lack of blocking diminishes the signal resolution obtained with PLBs as the functional surface, but it does not adversely affect functionality of the SAMs (data not shown). Antibodies labeled with fluorescent dyes are inherently more stable. Of such dyes, long wavelength dyes (such as AF647) have lower background fluorescence (18-20). Alexa Fluor dyes are suited for bioapplications because of their brightness, photostability, water solubility, and pH compatibility. AF647, in particular, shows relatively small change in the fluorescence spectra and intensity when conjugated to antibodies in contrast with other dyes such as Cy5 (20, 22). Following this reasoning, we selected AF647 for our experimental setup. As indicated in Figure 2, use of T84.66-NH-AF647 as the labeled reporter antibody results in a significant S/B with 100 pM of CEA. There is a 15-20% decrease in signal with repeat excitation (photobleaching) as is typically expected from such dyes. We also explored labeling T84.66 with AF647 in the hinge region. Labeling in this area is advantageous, since it directs the modification away from the antigen-binding region. This can be especially critical for larger reporters such as QDs. Mild reduction allowed for the generation of two half-antibody fragments containing free sulfhydryl groups in the hinge region. The fluorescent dye was then conjugated to the hinge region. When used as the detector for CEA, however, this material resulted in unusual signal enhancement (rather than photobleaching) with repeat excitation. It has been demonstrated that AF647 is more photostable and resistant to fluorescence quenching (at optimal degree of labeling) upon protein conjugation (20, 22). But the novel signal increase reported here is unprecedented. This antibody had 4 mol of AF647 per mole of protein, which is an optimal degree of labeling, according to the manufacturer. We have separated the labeled antibody from free AlexaFluor dye by gel filtration. In any case, we have also tested NSB associated with free AF647 in our assay and found no increase in fluorescence (data not shown). We speculate that, since multiple AF647 molecules are associated with a single antibody, some dye molecules are excited only after the fluorescence associated with others is quenched. This phenomenon, in combination to the relative photostability of AF647 dyes, could result in an increase in fluorescence. These hypotheses may explain why the increase in fluorescence is proportional to the concentration of the antigen, and hence the concentration of detector antibody in the system. Why this does not occur with T84.66-NH-AF647 (which also has ∼4 mol of the dye/mol of protein) is, however, unclear. A possible explanation involves the conformation of the antibody when labeled in the hinge region, which allows for fluorescence quenching and subsequent photon release. It is apparent that the unusual signal amplification allows for a better S/B with lower concentrations of the antigen. This feature may be advantageous in attempting to develop a point-of-care diagnostic for breast cancer, especially in low volume samples such as NAF. However, since the degree of excitation and extent of amplification are dependent on the antigen concentration, quantitation of this phenomenon is not easy. This is a disadvantage for cancer detection, because most tumor biomarkers are normally expressed in the body at basal concentrations, even without cancer present. Therefore, a test for cancer should be quantitative for accurate diagnosis. This labeling strategy does
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have potential for applications where a yes/no answer is adequate, e.g., infectious diseases like tuberculosis. No single biomarker is an accurate indicator of cancer. Therefore, an efficient diagnostic strategy should be able to detect multiple biomarkers in a single sample. To achieve this eventuality, we explored the use of QD labeled antibodies as a fluorescence detector in our assay system. In this manuscript, we evaluated both NH and SH labeling of antibodies. Both strategies allowed for the detection of CEA at a concentration of 100 pM, although the S/B was lower than that seen with T84.66-NH-AF647. The signal intensity measured with hinge labeling (Figure 4B) is lower than that seen with primary NH labeling (Figure 4A). However, the NSB is also lower. Therefore, the extrapolated S/B is comparable in both labeling strategies. Also, the observed signal is stable for over 2 min of excitation with the laser (Figure 4), a time over which signal from AF647 would be almost entirely photobleached. This photostability of core-shell quantum dots is a distinct advantage for multiplex detection as well, especially in attempting to engineer a multichannel sensor for simultaneous detection. It is also possible to envision an internal photostable standard (in lieu of streptavidin-AF647) that is used for both system functionality assessment and quantitation of antigen in unknown samples (unpublished data) (6). To conclude, we have improved a sandwich assay for CEA on our waveguide-based OB by incorporating better surfaces and photostable detectors. These modifications will enable our biosensor to function as a robust research tool to probe biological interactions or as a point-of-care diagnostic method to detect a variety of disease targets.
ACKNOWLEDGMENT The authors thank Karen Grace, Sohee Jeong, and Jennifer Martinez at the Los Alamos National Laboratory for help with waveguide instrumentation and helpful suggestions. This work was supported, in part, by a Los Alamos National Laboratory directed research (LDRD) grant.
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