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Biotechnol. Prog. 2001, 17, 543?552

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Application of an Antibody Biochip for p53 Detection and Cancer Diagnosis
Minoo Askari,?,? Jean Pierre Alarie,? Maria Moreno-Bondi,§ and Tuan Vo-Dinh*,?,?
Advanced Monitoring Development Group, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6101, Graduate School of Biomedical Sciences, University of Tennessee/Knoxville, Oak Ridge, Tennessee 37830-8026, and Departmento de Quimica Analitica, Universidad Complutense, 28040 Madrid, Spain

Detection of the p53 tumor suppressor gene is important in early cancer diagnostics because alterations in the gene have been associated with carcinogenic manifestations in several tissue types in humans. We have developed an antibody-based detection instrument, the biochip, to detect the presence of the anti-p53 antibody in human serum. The design of this highly integrated detector system is based on miniaturized phototransistors having multiple optical sensing elements, amplifiers, discriminators, and logic circuitry on an IC board. The system utilizes laser excitation and fluorescence signals to detect complex formation between the p53 monoclonal antibody and the p53 antigen. Recognition antibodies are immobilized on a nylon membrane platform and incubated in solutions containing antigens labeled with Cy5, a fluorescent cyanine dye. Subsequently, this membrane is placed on the detection platform of the biochip and fluorescence signal is induced using a 632.8-nm He-Ne laser. Using this immunobiochip, we have been able to detect binding of the p53 monoclonal antibody to the human p53 cancer protein in biological matrices. The performance of the integrated phototransistors and amplifier circuits of the biochip, previously evaluated through measurement of the signal output response for various concentrations of fluoresceinlabeled molecules, have illustrated the linearity of the microchip necessary for quantitative analysis. The design of this biochip permits sensitive, selective and direct measurements of a variety of antigen-antibody formations at very low concentrations. Furthermore, the acquisitions of the qualitative and quantitative results are accomplished rapidly, in about 15 min. These features demonstrate the potential of this antibody-based biochip for simple, rapid and early biomedical diagnostics of cancer.

Introduction
During past decades, the expansion of molecular biology has had a pivotal role in understanding the basis of cancer development and progression. Many key observations have been made concerning the genetic alterations associated with the disease, and carcinogenesis has been shown to be a stepwise process that occurs through mutations of cancer-related genes. These genes have been used to piece together a puzzle of regulatory systems that govern cell division and proliferation, as well as being involved in pathways leading to apoptosis (1-2). Recent advances in understanding of the pathogenesis of cancer have been helpful in addressing issues in diagnosis, prognosis and management of cancer patients with early detection being recognized as the key to better treatment strategies resulting in higher survival rates of patients. Accordingly, characterization of the molecular machinery whose alterations result in early preneoplastic transformation could potentially serve as early diagnostic marker (3). Tumor diagnostic markers are powerful means of
* To whom correspondence should be addressed: Advanced Monitoring Development Group, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6101, USA. Tel: 865-5746249. Fax: 865-576-7651. E-mail: vodinht@ornl.gov. ? Oak Ridge National Laboratory. ? University of Tennessee/Knoxville. § Universidad Complutense.
10.1021/bp010008s CCC: $20.00

obtaining information about cancers (4, 5). They possess utility for screening, detection, identification, prognosis and monitoring of different cancers (6-8). Of the presently available markers, the p53 tumor suppressor ranks high among those holding the greatest promise (9-11). The p53 protein functions as a negative regulator of cell growth, and alterations in the p53 gene lead to loss of this negative growth regulation, resulting in a more rapid cell proliferation (12-17). Extensive studies have systematically provided clinicopathologic and molecular support for association of abnormalities in the p53 gene with carcinogenesis in various human organs such as breast, endometrial, pancreatic, gastric, prostate, lung, skin, colorectal and esophageal cancers (18-28). In most instances, point mutations modify the conformation of the p53 gene, causing the p53 protein to accumulate in the nuclei of tumor cells and induce an immune response, which results in a higher concentration of p53 auto-antibodies in the serum of cancer patients. These statistically higher nuclear p53 levels and elevated levels of the circulating abnormal p53 auto-antibodies in the sera of the cancer patients have been associated with poor survival rates. In lieu of such observations, clinically valuable, positive correlation has been established between the concentration of the circulating auto-antibodies in the sera of patients and the disease stages in the affected individuals

? 2001 American Chemical Society and American Institute of Chemical Engineers Published on Web 04/11/2001

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(21, 29-31). These and countless other studies demonstrate the potential of the clinical evaluation of the serum p53-associated proteins to serve as highly specific tumor markers for cancer detection and monitoring of the disease progression in various cancer patients. Accordingly, development of a new molecular diagnostic technique with the ability to quantitatively detect the levels of p53 auto-antibodies in the human serum could serve as a major diagnostic tool with great significance in cancer detection and monitoring. In recent years, several optical sensor techniques have been developed for the direct monitoring of biomolecular recognition processes (32). Biosensors, in general, combine two important concepts that integrate affinity of specific biological molecules for each other with a sensing element for detection of this recognition process. The basic principle of an optical biosensor is to recognize this molecular recognition and to transform it into a detectable optical signal. A variety of optical biosensors used in enzyme -, immuno-, and DNA-based assays have been recently manufactured (33-45). Among the most recently developed diagnostic biosensors are single-target molecule detection with optical immuno-labels (33), fiberoptic-based protein detecting biosensors (34, 35), sol-gelbased biosensors for use in treatment (36) and planar array immuno-sensors for the detection of toxic agents (37). Our laboratory has recently developed a DNA biochip measuring fluorescence signals, based on integrated circuit microchip technology for use in medical diagnosis (38-40). The biochip used in the present study was based on photodiode array technology developed at Oak Ridge National Laboratory. A detailed description of the design of this integrated electro-optic system on integrated circuit (IC) microchips, including photodetector elements with amplifier circuitry, and their integration has been previously published (40). Very briefly, this highly integrated biochip system is a self-contained device the design of which is based on miniaturized phototransistors. This fluorescence-detecting device has multiple optical sensing elements, amplifiers, discriminators and logic circuitry that are fabricated on a single IC board. The performance of the microchips designed and fabricated for this study has previously been evaluated by measuring the fluorescence signal output response of the device for various concentrations of the fluoresceinlabeled DNA spotted onto the membrane probe. The results of the fluorescence calibration curve have illustrated the linearity of the microchip detectors and demonstrate the possibility for quantitative analysis (39, 40). The detection principal of most of the currently available microchip-biosensors is based on fluorescence signals being directed from the sample platform to an imaging system, such as a confocal microscope, equipped with external detectors. The conventional detecting systems generally incorporated in these setups include photomultipliers, charge-coupled devices (CCD), or charge injection devises (CID). Although the sample platforms themselves are quite small, the detection systems are relatively large, tabletop-size systems, suited for research laboratory applications. By exploiting the advantages afforded by the use of miniaturized phototransistors and the independently operating photodiodes, we have designed a truly integrated biochip system composed of probes, samplers, and detector as well as amplifier and logic circuitry on a compact IC board. The biochip itself is a 2 × 4 × 0.3 cm3 device incorporated into a setup that with all its components has 10 × 20 × 30 cm3 dimensions. This stable setup is designed for extended applications in

clinical offices, where it offers several advantages in size, performance, fabrication, analysis, and production cost due to its integrated optical-sensing microchip. Since the sample platform does not come in direct contact with the microchip itself, no regeneration process of the biochip itself is necessary for the next succession of detection. Additionally, the biochip design was based on complementary metaloxide-semiconductor (CMOS) technology, allowing design modifications (e.g., shape and size) of different electronic components for a wide variety of applications. The potential for implementing the CMOS technology for combining phototransistors and other microelectronics is another principal advantage of this sensor over the other 2D detectors. The extended application of this biochip is based on the antibody-antigen recognition process, which involves the identification of a specific target antibody, by its complementary sensing antigen probe (41-44). Biochips employing immunological reagent phases are particularly promising because they have the potential to perform highly selective and sensitive in situ measurements of a broad variety of immunological reactions in view of antibodies to an extensive range of antigens being commercially available or could be produced. Because they rely on the outstanding specificity of antibodyantigen recognition, antibody-based biochips offer a very high degree of accuracy and can be utilized to detect trace amounts of proteins in various biological matrices (45). In the present study we have further extended the capabilities of our biochip to detect the p53 protein in complex biological solutions. Using protein immunoassays coupled with laser instrumentation, we evaluated the performance of this immuno-biochip to potentially serve as a highly selective and ultrasensitive diagnostic tool. We set out to (a) verify the presence and quantify the concentration of p53 protein, (b) simultaneously detect multiple and independent immunoassays on the same sampling platform and (c) detect anti-p53 antibodies in human serum. This investigation determines the potential diagnostic value of this immuno-biochip for assessing the levels of the p53 protein, which can serve as a valuable tool for cancer diagnosis in clinical settings.

Instrumentation and Materials
Imaging Apparatus. Biochip. A WPI-PV830 pneumatic pico pump (World Precision Instruments, Sarasota, FL) was used for dispensing target antibody onto the membranes. Each membrane was secured to the nanopositioning stage of a Burleigh 6000 controller (Burleigh, model TSE-75, Fisher, NY) for two-dimensional (XY) movements. After production of the desired array of targets, the membrane was probed with the complementary labeled antigen, utilizing a Spectra-Physics (Eugene, OR) He-Ne laser (7 mW) as an excitation source. Corion filters (Franklin, MA) were used to filter out any scattered laser light from reaching the biochip’s detector and to spectroscopically isolate only the membrane fluorescence for these measurements. Other lenses used for the system alignment were supplied by Newport Optics (Irvine, CA) and a Protek 506 multimeter (Seoul, Korea) was used for fluorescent signal measurements. Photometric Charge-Coupled Device. For better evaluation of the results obtained using the biochip, images of the fluorescence membrane were also made with a liquid nitrogen cooled photometric charge-coupled device (CCD) using a Pentax 50-mm lens equipped with a Vivitar macro teleconverter (Japan). A Coherent Innova 70 (Palo Alto, Ca) krypton laser beam was employed as the excitation source, and a Raman holographic filter

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obtained from Kaiser Optical systems (San Jose, CA) and Corion filters (Franklin, MA) were used for alignment of the laser beam onto the CCD device. To determine the relative fluorescence intensities of the spots on the membrane, the system’s own software CCDOPS and the Spyglass software (Naperville, IL) analyzed the images produced by the CCD camera. Materials. Proteins. Monoclonal antibody to wildtype p53 raised in mouse ascites fluid was obtained from Sigma immunochemicals (St. Louis, MO). This monoclonal anti-p53 (mouse Ig2a isotype), which recognizes a denaturation-resistant epitope on the primate p53 nuclear protein (53 kD), has been derived from the BP53-12 hybridoma line and does not react with other cellular proteins. The stability of the antibody is estimated to range between 2 and 5 years, and the minimum working solution of 1:400 has been determined by indirect immunoperoxidase labeling of human tissues. Synthesized human p53 blocking peptide, used as the antigen against the p53 antibody, was obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Rabbit anti-goat IgG antibody and goat IgG antibody along with human serum from clotted male whole blood were purchased from Sigma immunochemicals (St. Louis, MO). Fluorescent Label. FluoroLink MAB Cy5 Labeling Kit was purchased from Amersham Life Science (Arlington Heights, IL). The water-soluble Cy5 dye is a bisfunctional NHS ester that binds to free amino groups and produces an intense fluorescent signal in the far red region of the spectrum (absorbance max ) 649 nm and emission max ) 670 nm). The kit is designed to label 1 mg of protein to a final molar dye/protein ratio between 4 and 12, assuming an average protein molecular weight of 155 kD. Membrane. Immunodyne ABC membrane was acquired from Pall Corporation (Port Washington, NY). This is a nylon membrane the surface of which has been modified with reactive groups to covalently bind the amino group of proteins. Chemical Reagents. All other chemicals were of analytical reagent grade and were purchased from Sigma Chemical Co. (St. Louis, MO). Water used for solution preparation was distilled and deionized using the Millipore’s Milli-Q system (Bedford, MA).

Experimental Procedures
Cy5 Labeling of the Antigens. One milligram of each protein, the p53 human blocking peptide and the goat IgG antibody, were dissolved in 1 mL solution of 0.1 M sodium carbonate bicarbonate buffer (pH 9.3) and transferred to vials containing 100 nM of the Cy5 labeling dye. After thorough mixing, the reaction vials were incubated at room temperature for 30 min with additional mixing every 10 min. The labeled proteins were subsequently separated from the unconjugated dye through Sephadex G-50 gel permeation columns (packed column length ) 12 cm) equilibrated with phosphatebuffered saline (PBS) (pH 7.2). The faster moving bands corresponding to the Cy5-labeled proteins were collected. The degree of protein labeling was estimated by calculating the ratio of the molar concentrations of dye to that of the protein. The final value of dye/protein ratio was estimated at 1 dye molecule per molecule of the p53 human blocking peptide and 6 dye molecules per molecule of the goat IgG antibody. Altering the starting concentration of the protein and the reaction pH affected the labeling efficiency of this conjugation reaction. The optimal labeling generally occurred at pH 9.3 and in-

creasing the protein concentration enhanced the labeling efficiencies. The final concentrations of the proteins in the collected fractions were analyzed by measuring the absorbance spectra of the samples at 280 nm. Antibody Microarray Immobilization and Probing. To ensure optimal contact and maximum detection, the following procedure was used for antibody immobilization and stabilization on the surface of the membranes. Immunodyne ABC membranes cut to approximately 2 × 4 cm2 were used as the sampling platform. For each experiment a cut membrane was affixed on the stage of a nanopositioner capable of twodimensional (XY) movements. Using a pneumatic picopump, having a 100-?m i.d. capillary-dispensing tip, a volume of 0.1 ?L of the selected target antibody (500 ?g/ mL in PBS) was deposited on the membrane to generate an individual spot. The translational stage was then moved so as to achieve the desired array of target antibodies. Specifically, a 4 × 4 microarray spot arrangement was dispensed in a 0.8-cm2 area of the membrane with a constant distance of 1.1 mm, center to center, maintained between the spots in each row and column. This arrangement allowed the position of target antibody spots on the membrane to exactly match the spacing and arrangement of the sensing array on the biochip detection platform. The 4 × 4 array configuration in these experiments, with each specific target antibody being dispensed four times in a row, provided quadruplicate measurements of the same sample for data analysis. After generation of the desired microarray, antibodies were allowed to stabilize on the membrane for 5 min at 25 °C under approximately 50% relative humidity. The membranes were then blocked in 4 mL of the blocking solution, containing 2 mg of bovine serum albumin (BSA) in 1 mL of phosphate buffered saline (PBS). After 30 min of the blocking procedure, the membranes were rinsed three times in PBS solution and further incubated for 1 h at room temperature in the appropriate labeled probe solution (250 ?g/mL of either Cy5-labeled p53 blocking peptide or Cy5-labeled goat IgG in PBS). The membranes were then washed two times in 10 mL of washing solution (0.1% Triton X-100 in PBS v/v) for 1 min each at room temperature to reduce the background fluorescence signals. This step was followed by another rinse in 10 mL of PBS for 1 min and storage in 4 °C PBS until signal measurement. Signal Detection and Measurement Systems. Laser-Based Biochip System. The integrated electro-optic system of the biochip has been previously described (40). A schematic diagram of the biochip concept is presented in Figure 1. A basic biochip includes the following components: (1) excitation light source with related optics, (2) a bioprobe, (3) a sampling element with sample platform and delivery system, (4) an optical detector with associated optics and dispersive device, and (5) a signal amplification/treatment system. In these experiments, the emitted light from a 6.4-mW He-Ne laser (632.8 nm) was used as an excitation source. The laser light was passed through a laser band-pass filter, a collumating lens, and a 4 × 4 optical diffractive element and subsequently focused onto the active area of the membrane, where the Cy5-labeled antigen probes had adhered to their corresponding immobilized antibodies. The nylon membrane was placed on a microscope slide directly above the phototransistor detection element. The fluorescence signals from the excited Cy5-labeled antigen probes were then passed through a graded index of refraction (GRIN) lens. A 650-nm band-pass filter was situated between the detector elements and the sample

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Figure 1. Schematic diagram of a biochip concept. Light from a 6.4-mW He-Ne laser (632.8 nm) was transmitted through a laser band-pass filter, a collimating lens, and a 4 × 4 optical diffractive element and was focused onto sample spots situated directly above the phototransistor detection elements. The 4 × 4 sensing array is composed of 16 individual photodiodes arranged in a square with 900-?m edges and 1-mm center-to-center spacing between them. Standard CMOS process was used for fabrication of the photodiodes and the associated electronic circuitry. A GRIN lens and a 650 ( 10 nm band-pass filter was situated between the detector elements and the sample substrate to isolate the fluorescence of interest. The photocurrent of each sensing element of the amplifier/phototransistor microchip was transmitted to a digital voltmeter linked to a multimeter for data recording.

substrate to isolate the resulting fluorescence signal from the laser line and direct it to the onboard detectors. Direct quantification of the fluorescence intensity of the signals was performed by the integrated components of the biochip and the photocurrent of each sensing element of the amplifier/phototransistor microchip was transmitted to a digital multimeter for instant data recording. In this study, we used near-infrared (NIR), 700- to 1000-nm, fluorescent dye for labeling our immune probes, because measurements in the NIR have less interference from background fluorescence of the membrane. Another advantage with NIR measurements is the availability of inexpensive lasers with emission lines in the red and NIR regions. CCD Multichannel Detection System. As a comparative method for detection and visualization of the induced fluorescence signals, a liquid nitrogen-cooled photometric charge-coupled device (CCD) was also employed. The CCD setup consisted of a krypton laser beam (647 nm) for the excitation source plus multiprobe waveguide, optical filters, lenses, and a CCD detector. The active membrane was placed on a clean microscope slide and positioned on the imaging platform of the CCD camera. The laser beam was transmitted through a 600?m diameter fiber and passed through a 670-nm-long pass to block unwanted laser lines and remove background fluorescence emission from the optical fiber. This beam was then focused on the active area of the membrane for generation of the fluorescence signals. To further remove any remaining laser light, the fluorescence signal was then passed through a 514.5-nm Raman holographic filter. Employing a 1:250-mm lens and a 2×

macrofocusing teleconverter, the signal was then focused onto the surface of a CCD detector and a two-dimensional image was captured.

Results and Discussion
Evaluation of Antibody-Antigen Conjugation. Increases in the concentration of serum p53 autoantibodies, as a result of its overexpression due to the p53 gene mutation, appear as a regular occurrence in the progression of cancers of many organs. Therefore, analysis of this increase in the serum could prove extremely valuable for the diagnosis of the disease in affected individuals and for the selection of appropriate treatment regimens (18-28). The aim of this study was to evaluate the capabilities of our immuno-biochip to not only detect the presence of the anti-p53 antibody but to quantitatively determine the concentrations of this protein in biological matrices. In these experiments the different IgG proteins, the rabbit anti-goat IgG and the Cy5labeled goat IgG were used as the control proteins because of their prevalence and ubiquitous nature in the serum of individuals with or without a diseased condition. The operation of this biochip is based on the wellestablished processes of antibody-antigen recognition and the widely acknowledged suitability of the tumor suppressor p53gene as a potential prognostic marker with predictive value for cancer incidence and progression. The procedure was employed to detect complex formation between the anti-p53 monoclonal antibody and its complementary Cy5-labeled p53 blocking peptide using the integrated electro-optic microchip.

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Figure 2. CCD detection of p53 protein. The CCD system captured the 2D image (a) and the 3D image (b) of the antibody reaction in the presence of Cy5-labeled p53 antigen. The two channels showed strong signals corresponding to the reference channels used for alignment. Of the sample rows, only the top two rows corresponding to the immobilized p53 antibody displayed a fluorescence signal; there is no detectable signal produced from the rabbit anti-goat IgG spots.

Figure 3. Biochip fluorescent signal measurement and analysis for detection of p53 protein. Fluorescent signals were detected from the p53 immobilized antibody rows (rows 1 and 2). No significant fluorescence was observed from the negative control group, the rabbit anti-goat antibody spots on row 3. Signals measured using the biochip corresponded to the results obtained from the CCD imaging study.

The CCD instrument setup was incorporated into this study for two reasons. First, since the sensing elements of the photodiode biochip provided signal intensity and not a signal profile, using a CCD detection system we could record the fluorescent response of the sample spots on the platform. This system enhanced our visualization capability for detection of (a) interactions between the immunological probes and their specific target antibodies, (b) the degree of cross-reactivity of the fluorescently labeled probes with the two immobilized target antibodies and (c) the degree of nonspecific binding and the background fluorescence of the membrane. The second reason for using the CCD setup was to provide us with an alternative fluorescence detection technique to compare with the signal information obtained using the biochip detection system. Assay for the Detection of p53-Associated Proteins. The development and evaluation of the target antibody microarray are important elements of the biochip function. The spot arrangement should be created in a manner as to superimpose the pattern of the excitation-induced fluorescence signals directly onto the array of the detector elements, located on the detection platform of the biochip. In this experiment, a 4 × 4 array of microspots was generated on the sample substrate. First, the anti-p53 antibody was dispensed as four target spots in a row, on the top two rows of the array. As a control, four spots of the rabbit anti-goat IgG was dispensed on the third row of the same array. To aid in the alignment of the microarray, Cy5-labeled goat IgG was spotted on the first and fourth spot positions of the fourth row of the array. The membrane was then blocked and probed with the Cy5-labeled p53 blocking peptide. The CCD detection system was used first to evaluate the fluorescence signals generated from the spots on the membrane by providing a 2D image of the sample array. Figure 2 shows the CCD-captured 2D image of the 4 × 4 array after incubation with the Cy5-labeled p53 blocking

peptide. The two channels showing strong signals correspond to the reference channels, spotted with Cy5labeled IgG and used for calibration and alignment of the system. As demonstrated, only the top two rows (rows 1 and 2), corresponding to the immobilized anti-p53 antibody spots, displayed fluorescence signal. This verified complex formation between the anti-p53 antibody and its complementary antigen. Moreover, since there was no detectable signal produced from our control group, the rabbit anti-goat IgG spots deposited on the third row of the same membrane, the specificity and the selectivity of the anti-p53 antibody and the p53 blocking peptide was established. This lack of fluorescence signal from the rabbit anti-goat IgG spots demonstrated the absence of cross reactivity of the Cy5-labeled p53 blocking peptide with the rabbit anti-goat IgG target antibody, present on the same membrane and in the same macro-environment. Next, the biochip analysis of the induced fluorescence signal was carried out by first inserting the abovementioned membrane onto the detection platform. Fluorescence signals from each spot were measured individually, and the photocurrent of the sensing surface was readily available from the on-board logic circuitry of the biochip without the need for any external electronic interface system or signal amplification devices. Once all of the spot measurements were completed, the fluorescence/background ratios (relative fluorescence) were plotted. Figure 3 shows the response obtained after incubation of the specified membrane with the Cy5-labeled p53 blocking peptide. Significantly higher than background fluorescence signals were observed from the anti-p53 antibody spots immobilized on rows 1 and 2. The negative control group, the rabbit anti-goat antibody spots, on row 3 did not exhibit any significant fluorescence signals. The results of this experiment established three facts. First and foremost, the fluorescence detection capability of the biochip to detect complex formation between the anti-p53 antibody and its complementary Cy5-labeled p53 blocking peptide was illustrated. The biochipobtained information corroborated extensively with the signals achieved using the CCD imaging system (Figure

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Figure 4. Selectivity study. The CCD-captured 2D visual image (a) and its 3D plot (b) exhibit fluorescence only from the rabbit anti-goat IgG row (row 3) and the reference channels (row 4) after addition of Cy5-labeled goat IgG. No fluorescence signal was observed from the top two rows (rows 1 and 2), where the p53 antibody spots were dispensed, confirming absence of cross reactivity between p53 antibody and Cy5-labeled goat IgG protein.

Figure 5. Selectivity study. The biochip fluorescence signal measurements fully corresponded to the CCD imaging results. Fluorescence signals were only detected from the IgG and the reference rows, displaying no cross reactivity between the p53 and the goat IgG antibodies and antigens.

2). Second, the specific affinity and the selectivity of the Cy5-labeled blocking peptide to identify its target molecule, the anti-p53 antibody, were demonstrated. Last, the absence of cross reactivity between the Cy5-labeled p53 blocking peptide and the rabbit anti-goat IgG, which were spotted on the same membrane, was illustrated. These results were highly significant since satisfactory performance of any new detection technology requires that the device only detects the molecule of interest and have minimal signal contributions from other components present in the system. Selectivity Study. Induced fluorescence responses were monitored after incubating a second membrane, spotted with anti-p53 antibodies in the top two rows, rabbit anti-goat IgG on the third row, and the alignment spots on the fourth row of a 4 × 4 array, in a solution containing Cy5-labeled goat IgG. The CCD-captured 2D image Figure 4 exhibits fluorescence signal produced only from the rabbit anti-goat IgG row (row 3) in response to complex formation with the Cy5-labeled goat IgG in the probe solution. No fluorescence signals were observed from the anti-p53 antibody spots on rows 1 and 2. The visual observation of this membrane by CCD did not demonstrate a statistically significant level of cross reactivity between the immobilized anti-p53 antibody and the Cy5-labeled goat IgG protein. The signal measurements performed by the biochip (Figure 5) corresponded well to the results of the CCD imaging study. Fluorescence signals were only detected from the rabbit antigoat IgG row and the alignment spots. This assay also displayed no detectable cross-reactivity between the uncomplimentary antibody and antigens, and further confirmed the absence of false positive results. Study of Multi-Target Detection. In diagnosis of human diseases, it is at times desirable to obtain information on the expression status of the different proteins that are involved in manifestation of the disease. Specific examples include detection of different biomarkers and cancer antigens are associated with carcinogesis in different human organs such as breast, lung and

pancreatic cancer (46-50). Therefore, simultaneous detection of multiple protein molecules would be a valuable feature in a detection technology to be used in a clinical or research laboratory. We evaluated the capability of the biochip to concurrently detect both of our target antibodies, the anti-p53 antibody and the rabbit anti-goat IgG. A multiarray containing sample spots of both proteins was produced for use in this experiment. Solutions containing anti-p53 antibody and anti-goat IgG were dispensed on rows 1 and 4, respectively, of this array on a membrane. The membrane was subsequently blocked and incubated in a solution containing both the Cy5labeled p53 blocking peptide and the Cy5-labeled goat IgG probes. The results of the study illustrated in Figure 6 show fluorescence signals detected from both the rabbit anti-goat IgG spots dispensed on the first and the antip53 antibody spots dispensed on the fourth rows of the microarray. The biochip device allowed simultaneous detection of both immobilized target antibodies on the same membrane when using a probe solution containing more than one Cy5-labeled antigen probes each complementary to each of the target antibodies. These results demonstrated the potential of the biochip for use with many different complementary antibody/antigen complexes. This valuable feature of the biochip should provide for a more comprehensive and versatile clinical diagnostic technology. Study of Nonspecific Binding Effect. To accomplish a comprehensive evaluation of the biochip system, it was important to ascertain the level of fluorescence inherent to the biochip system itself and from nonspecific adsorption of the Cy5-conjugated probes. A 2 × 4 cm2 piece of membrane was cut and incubated in the BSA-containing blocking solution. As previously described, this membrane was incubated in a solution of the Cy5-labeled goat IgG. After the required incubation period, the membrane was washed and placed onto the detection platform of the biochip. The combined sum of the inherent fluorescence signals contributed from the biochip and that of the membrane was much lower when compared to the fluorescence signals induced through laser excitation of the labeled protein complexes (data not shown). Exposure of membranes to the Cy5-labeled probes resulted in only a 1-2% increase in the background fluorescence signal

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Figure 6. Study of multi-target detection. The biochip device permits simultaneous detection of immobilized p53 antibody and goat IgG target antibodies spotted on the same membrane.

Figure 7. Quantitative analysis. Biochip capability for quantitative analysis of different concentrations of the p53 protein is demonstrated. The relative fluorescence signal detected by the biochip increased in response to an increase in concentration of immobilized target antibody.

when compared to control membranes incubated in solutions containing no Cy5-labeled probes. Additionally, the fluorescence signals were extremely uniform in nature providing for an excellent baseline for fluorescence signal collection using this biochip. Quantitative Analysis. To take advantage of the well-established correlation between the concentration of p53 serum proteins and the disease stage in cancer patients, it is crucial for the biochip to have the capability for quantitative analysis of the protein of interest. To establish the ability of the biochip to measure different concentrations of the p53 target antibody, a concentration curve assay was carried out. For this assay, a membrane was spotted with solutions containing different concentrations of the anti-p53 antibody, ranging between 14 and 450 ng/mL, and incubated in a probe solution of Cy5labeled p53 blocking peptide. Figure 7 demonstrates the magnitude of the relative fluorescence signal obtained by the biochip, in response to changing the concentration of the immobilized target antibodies on the membrane. The amount of fluorescence signal increased with the increase in the concentration of the target anti-p53

antibody. These results point to the sensitivity and the effectiveness of the biochip technology for quantitative analysis of tumor markers. Analysis of p53 Target Protein in Human Serum. The p53 antibodies are a recently discovered serological parameter in individuals with malignancies. Their occurrence has been described in various types of cancer, and several studies in cancer patient groups have indicated its prognostic value (51-56). In most instances, point mutations modify the conformation of the p53, causing overexpression and accumulation of the p53 protein in tumor cells as a result of increased protein stability. Production of such abnormal p53 proteins with prolonged half-lives allows their detection by antibodies, leading to the development of serum p53 auto-antibodies. The concentration of p53 protein in the serum of patients varies in carcinogenesis of different organs. Antibody levels ranging from a few hundred to 9 × 106 arbitrary units/L in serum of patients with ovarian carcinoma (51), and 4.5 mg/mL of soluble p53 protein in the blood of colon cancer patients has been observed (56). These observations suggested that detection of serum anti-p53 antibody

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Figure 8. Analysis of p53 target protein in human serum. Increase in signal output in response to the increase in the concentration of the p53 antibody in serum is illustrated. The results illustrate the linearity of the microchip detector with respect to increase in the concentration of the target antibody.

quantitative analysis of the p53 protein, cancer tumor marker, both in saline solution and in human serum. This system offers numerous capabilities as a potential diagnostic tool for detection of p53-associated proteins in the serum of cancer patients. With its multichannel capability, the immuno-biochip is a sensitive and selective system that will allow rapid, simultaneous detection of different biological targets using various antibody probes. The fusion of microelectronics, CMOS technology and molecular biology has created this new technology, which provides an efficient and cost-effective tool in molecular diagnostics. The biochip is suited for integration into fully automated systems, thus providing the basis for automation of molecular diagnostics with potential for high sample throughput. This compact system is a user-friendly, inexpensive device amenable to on-site use at doctors’ offices. These features demonstrate the future potential of this biochip for cancer detection in clinical settings.

could be a new approach not only for the investigation of the status of the p53 gene in tumor cells but also for the disease prognosis in the cancer patient and a criterion for choice of treatment strategy. Therefore, in this experiment we set out to evaluate the performance of the biochip to analyze and quantify the levels of p53 antibodies mixed in human serum. To establish the ability of the biochip to quantitatively determine p53 antibody levels in the human serum, different amounts of the anti-p53 antibody mixed in human serum were prepared. The concentrations of the p53 antibody in the human serum were in accordance with the published concentrations of the p53 autoantibodies observed in the serum of patients with cancers of various organs. A membrane was spotted with different concentrations of the p53 target antibody in serum, ranging between 0 and 3.00 mg/mL, and then incubated in a solution of Cy5-labeled p53 blocking peptide. Figure 8 demonstrates the increase in signal output, in response to the increase in the concentration of the p53 antibody in serum immobilized on the nylon membrane. The results illustrate the linearity of the microchip detector with respect to increase in the concentration of the target antibody. This demonstrated the possibility for the biochip to be a sensitive and effective technology for quantitative analysis where information on concentrations of tumor markers in serum is desirable.

Acknowledgment
This research is jointly sponsored by the Office of Biological and Environmental Research, U.S. Department of Energy, under contract DE-AC05-00OR22725 with UT-Battelle, LLC., and by the ORNL Laboratory Directed Research and Development Program (Advanced Nanosystems).

References and Notes
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Conclusion
The aim of the present study was to develop a rapid, simple and practical approach for cancer detection. We have described an approach based on the application and further development of a unique technology, the biochip, utilizing the excellent sensitivity of fluorescence detection in conjunction with the biological specificity of antibodyantigen recognition and interaction. The biochip technology described in this work is a multiarray optical biosensor that is based on integrating the photosensing microchip systems, signal amplifiers and data treatment technologies. Our results indicated that fluorescently labeled biological proteins could be reliably detected using membrane-immobilized antibodies on the biochip-sampling platform. This biochip not only allowed detection of individual antibody targets but also provided for simultaneous detection of multiple antibodies immobilized on the same membrane. Another feature of this immuno-chip was its ability to detect molecules in complex biological matrices. Our investigations demonstrated the ability of this biochip for detection and

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Accepted for publication January 16, 2001. BP010008S


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