Biomedical Microdevices 7:2, 143–146, 2005 C 2005 Springer Science + Business Media, Inc. Manufactured in The Netherlands.
Parallel Detection and Quantification Using Nine Immunoassays in a Protein Microarray for Drug from Serum Samples Hongwu Du,1,2,4 Weiping Yang,2 Wanli Xing,1,2,4,5 Yao Su,3 and Jing Cheng1,2,4,5,∗ 1 Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, People’s Republic of China 2 National Engineering Research Center for Beijing Biochip Technology, Beijing 102206, People’s Republic of China 3 Beijing Science Technology and Management College, Beijing 100086, People’s Republic of China 4 Medical System Biology Research Center, School of Medicine, Tsinghua University, Beijing 100084, People’s Republic of China 5 State Key Laboratory of Biomembrane and Membrane Biotechnology, Beijing 100084, People’s Republic of China E-mail:
[email protected]
Abstract. A protein microarray system for detection and quantification of nine prohibited drugs in serum is described. Chemically modified slides were chosen as the microarray substrates because of their suitable for drug-BSA printing. The developed protein microarray was able to preserve the biological function of the haptens, when immobilized on the microarray surface and demonstrated binding with their corresponding antibodies. The microarray could also be used for quantitative analysis when mouse IgG was chosen as an internal control for data processing. There was no qualitative difference between the results obtained using the protein microarray and ELISA. The protein microarray technology should be applicable to performing, simultaneously, large scale screening tests for many different analytes in serum. Key Words. nine Immunoassays, serum, protein microarray
1. Introduction Unlawful drug use among adolescents is a major public health problem. Use of amphetamine, morphine, methadone, methamphetamine, PCP, and other stimulants or narcotics are irresistible among adolescents and such misuse has risen significantly in the recent years (Jenkins, 2002). During the same period, adolescents have reported less perceived dangers in using prohibited drugs and have become less disapproving of individuals engaged in this activity (Laure et al., 2003). These trends are alarming, given the numerous acute and long-term adverse consequences associated with drug misuse (Forbes, 1985). Simple ethic education would not appear to prevent the abuse of drugs and screening for drugs of abuse in biological fluids has been proposed as an effective way to combat this problem. Separation assays (gas chromatography/mass spectrometry, GC/MS) and Immunology assay (ELISA) have been successfully employed for the
detection of drugs abuse in the clinical laboratory (Wilson, 1988), but there are some limitations for large scale screening. Immunoassay has been traditionally performed on an individual sample in respect of a single analyte while separation assay can identify minute amounts of analyte, its use as a screening tool is limited since it requires expensive instrumentation and time-consuming procedures of sample preparation. No single extraction procedure will extract all drugs from biological fluids and consequently GC-MS methods are usually reserved as a confirmation tool for samples previously identified by an established screening method (Duntas, 2003). Microarray technology is one of the fastest growing fields in the analytical sciences (Cheng et al., 1998; Van Ingen, 2002). It is becoming a method of choice for the simultaneous and high throughput measurement of multiple analytes using non-porous and porous solid supports combined with fluorescence-based detection (Lander, 1999; Stahler, 2002). Microarray immunoassays promise to revolutionize both clinical diagnosis (Cahill, 2001) and drug testing (Birchard, 2000) in situations where there needs to be simultaneous detection of multiple analytes in a single assay. Previous work has used a conjugated hapten microarray to screen for steroids in urine samples (Du et al., 2004) but for the present work a contact microarray printer was used to develop chips. BSA conjugated drugs were immobilized at discrete locations on the surface of home made aldehyde-modified glass slides. Competitive immunoassays were then performed and the bound anti-drug antibodies subsequently detected with a fluorescent-labeled * Corresponding
author.
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secondary antibody. The signal pattern of fluorescence was imaged using a confocal scanner. The fluorescence signals from individual test regions were then processed with special digital software. Ten arrays can be imaged simultaneously. Former studies (Du et al., 2005) focused on urinalysis but this study describes a protein microarray system for the detection and quantification of the prohibited drugs in serum.
2. Materials and Methods 2.1. Materials Ten conjugates (Amphetamine-BSA, Barbiturate-BSA, benzoylecgonine-BSA, Digoxin-BSA, Methamphetamine-BSA, Methadone-BSA, Morphine-BSA, Phencyclidine (PCP)-BTG, Tricyclic antidepressants (TCA)-BSA, Theophylline-BSA), and their antibodies were purchased from Fitzgerald (Concord, MA). Cy3 goat anti-mouse mAb (1 mg/mL) were purchased from Amersham Pharmacia (Buckinghamshire, England). Unless otherwise specified, all chemicals used were of reagent grade. 2.2. Preparation of aldehyde modified substrates The smooth and clean glass slides were immersed into chromium acid for a minimum of 6 hours, after which they were first rinsed with tap water and then with distilled water, and finally treated with 2 M sodium hydroxide for 30 minutes. After rinsing the slides were then dipped into 4 M hydrochloric acid for 30 minutes, dried and then shaken and then immersed in a 4% 3Glycidoxypropyltrimethoxysilane solution for 8 hours. After washing the surface of the slides sequentially with toluene, acetone and water. Finally the slides were immersed into 50 mM NaIO4 for one hour to produce the alderhyde active surface. 2.3. Preparation of conjugated hapten microarray Ten conjugated haptens as well as controls (mouse IgG, blank and BSA) were printed on the aldehyde-modified slides by a high-speed robotic station (PixSys 5500, Cartesian Technologies, Irvine, CA). Samples were transferred from the 384-microtiter plates to the glass slides using stainless steel pins (SMP3, TeleChem International, Inc. Sunnyvale, CA). The microarray consisted of a 9 × 6 matrix and each material was printed in triplicate (Figure 1). The mouse IgG acted as a reference and a normalization index for the data evaluation. This helps in the comparison of the microarray variance between batches. All printed substances were dissolved in printing solution (50 mM phosphate-buffered saline, 40% aqueous glycerol). Cy5BSA (5 µg/mL) was added to the printing buffer to help assess an equable distribution between the different spots.
Fig. 1. Sample matrix of the microarray. PC, positive control, mouse IgG (1 mg/mL); Blank, printing buffer; NC, negative control, BSA solution (1 mg/mL); others are BSA-drugs (0.5 mg/mL).
16 Printings were performed in a chamber at room temperature and 50% humidity. Such conjugated hapten microarrays can be stored at room temperature for up to 3 months. 2.4. Competitive-immunochemical microarray assay A molded polyester frame was attached to the substrate to partition ten arrays on the protein microarray surface. The microarrays were immersed into the blocking solution (10% sheep serum and 90% PBS, v/v) at 37◦ C for 30 min to block the unused sites and then rinsed three times with PBST (phosphate buffered saline). The protein microarray was then maintained at 37◦ C in a humidified chamber for 30 min. Next, the protein microarray was rinsed three times with PBST and the secondary antibody (Cy3labelled goat anti mouse IgG) was applied to the protein microarray and incubated at 37◦ C for 30 min. After rinsing with water for 10 minutes, the microarray was ready for image scanning using a commercial confocal scanner (ScanArray Express, Perkin Elmer Life and Analytical Sciences, Boston, MA) or a homemade scanner (LuxScan10K, CapitalBio, Beijing, China). The analogue fluorescence signal was converted into a digital signal by data analysis software (GenePix Pro 4.0, Axon Instruments).
3. Results and Discussion 3.1. The function of antibody mixture The developed protein microarray system was seen to retain the biological function of the haptens, when immobilized on the microarray surface and these were capable of binding with their corresponding antibodies. By choosing a suitable titer for the different antibodies to meet established criteria (Fluorescence between 10000–30000, PMT = 60, LASER POWER = 70) a first antibody mixture was formed, which was used in the microarray. The result was shown in Figure 2. 3.2. Detection of nine drugs In a prototype, aldehyde modified slides were used as the microarray support and the microarray surface was
Parallel Detection and Quantification Using Nine Immunoassays in a Protein Microarray
Fig. 2. Array image after the first antibody mixture was used.
chemically modified to facilitate the covalent binding of protein molecule at the designed location. On each slide, ten arrays were printed and each array contained three dots for different analytes. This means that one sample can be tested in one array for a variety of analytes. Proteins immobilized on aldehyde treated slides are very stable.
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Drug-BSA conjugates were immobilized on glass slide as a model system to test for drugs of abuse. Antibodies against corresponding drugs are used as recognition moieties. Competitive immunoassay for drug detection was adopted. The captured antibodies on the glass surface were added to the test samples. The immunoreactions took place in a reaction chamber for an hour and the free drugs were washed thoroughly with washing buffer (PBST). Fluorescence-labeled secondary antibodies were added as reporter molecules. If the tested sample contains any banned drugs, the fluorescence signal will decrease in the corresponding location. Signal decrease is proportional to the amount of drug molecules present in the tested samples. In principle, the fluorescence signal at the corresponding location is decreased when a tested substance is in the sample because the drug in the sample competes with drug immobilized on the protein microarray (BSA-drug) for the antidrug antibody. The response to the addition of 1 µg/mL of free drug in the presence of the first antibody mixture to different array is illustrated in Figure 3.
Fig. 3. Analysis for nine individual drugs using protein microarrays. The frame in red displays the expected binding of the antibody to its counterpart triplicate test spots. Signal decrease is proportional to the abundance of free drugs in the sample. (a) methamphetamine; (b) theophylline; (c) barbiturate; (d) amphetamine; (e) methadone; (f) morphine; (g) PCP; (h) TCA; (i) digoxin.
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of the antibodies and ranged from 0.2 ng/mL for morphine to 19 ng/mL for methadone. Within the linear range of measurement, the decrease in fluorescence signal was proportional to the amount of drug in the sample. Therefore, this method can be used for both qualitative and quantitative determination of these substances in a sample. Standard curves for the 9 drugs measured using the protein microarray are shown in Figure 4. The correlation coefficient (R 2 ) for the protein microarray was higher than 0.99.
4. Conclusion This protein microarray is capable of screening for multidrugs in serum samples and can report all the questionable drugs at the same time. It has the potential to become an optional method for screening drugs prohibited by the governments. With further optimization, such microarray systems may evolve into an acceptable screening method to test all prohibited drugs. Its performance is superior to that of ELISA.
Acknowledgment We would like to thank Prof. Brian Caddy for critical comments and suggestions on this manuscript. This work was funded by the Beijing Natural Science Foundation of China (No. H010210640121) and the National Hi-Tech Program of China (No. 2002AA2Z2011).
Fig. 4. Standard curves for 9 drugs measured using the protein microarray.
3.3. Quantitative analysis In addition to serving as a qualitative screening tool for large number of samples, the protein microarray can also be used for quantitative analysis. Antibody affinity can range from 10−6 to 10−12 mol/L and the sensitivity of this system relies mostly on the antibody affinity. An antibody with high affinity (>10−9 ) to the protein microarray was used whenever possible. An average sensitivity for different drugs was of the order of nanograms. This sensitivity level is sufficient for the detection of most drugs under doping control. The detection limit varied with each substance due to the different properties
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