A Universal Nucleic Acid Sequence Biosensor with Nanomolar

A quantitative universal biosensor was developed on the basis of olignucleotide sandwich hybridization for the rapid (30 min total assay time) and hig...
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Anal. Chem. 2004, 76, 888-894

A Universal Nucleic Acid Sequence Biosensor with Nanomolar Detection Limits Antje J. Baeumner,* Jennifer Pretz, and Shirley Fang

Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14853-5701

A quantitative universal biosensor was developed on the basis of olignucleotide sandwich hybridization for the rapid (30 min total assay time) and highly sensitive (1 nM) detection of specific nucleic acid sequences. The biosensor consists of a universal membrane and a universal dye-entrapping liposomal nanovesicle. Two oligonucleotides, a reporter and a capture probe that can hybridize specifically with the target nucleic acid sequence, can be coupled to the universal biosensor components within a 10-min incubation period, thus converting it into a specific assay. The liposomal nanovesicles bear a generic oligonucleotide sequence on their outer surface. The reporter probes consist of two parts: the 3′ end is complementary to the generic liposomal oligonucleotide, and the 5′ end is complementary to the target sequence. Streptavidin is immobilized in the detection zone of the universal membranes. The capture probes are biotinylated at the 5′ end and are complementary to another segment in the target sequence. Thus, by incubating the liposomal nanovesicles with the reporter probes, the target sequence, and the capture probes in a hybridization buffer for 20 min, a sandwich complex is formed. The mixture is applied to the membrane, migrates along the strip, and is captured in the detection zone via streptavidin-biotin binding. The biosensor assay was optimized with respect to hybridization conditions, concentrations of all components, and length of the generic probe. It was tested using synthetic DNA sequences and authentic RNA sequences isolated and amplified using nucleic acid sequence-based amplification (NASBA) from Escherichia coli, Bacillus anthracis, and Cryptosporidium parvum. Dose-response curves were carried out using a portable reflectometer for the instantaneous quantification of liposomal nanovesicles in the detection zone. Limits of detection of 1 fmol per assay (1 nM) and dynamic ranges between 1 fmol and at least 750 fmol (1750 nM) were obtained. The universal biosensors were compared to specific RNA biosensors developed earlier and were found to match or exceed their performance characteristics. In addition, no changes to hybridization conditions were required when switching to the detection of a new target sequence or when using actual nucleic acid sequence-based amplified RNA sequences. Therefore, the * Corresponding author. Telephone: 607-255-5433. Fax: 607-255-4080. Email: [email protected].

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universal biosensor described is an excellent tool for use in laboratories or at test sites for rapidly investigating and quantifying any nucleic acid sequence of interest. The detection of DNA and RNA molecules has become of increasing importance in recent years. Nucleic acid molecules are used to diagnose diseases; to detect pathogenic organisms present in environmental, food or clinical samples; and are screened in high throughput systems, such as microarrays for genomics and functional genomics analyses. Because of the inherent nature of DNA, any self-replicating organism can be discriminated from another on the basis of nucleic acid sequences unique to that particular organism. Various strategies to identify unique DNA sequences have been explored. Standard lab methods of detection rely on procedures such as Northern and Southern blotting in which the nucleic acid fragments are first separated according to size via agarose gel electrophoresis and subsequently transferred via capillary action onto a membrane for detection using radiolabeled, fluorescent, or chemiluminescent probes.1 These methods are labo-r and time-intensive, since detection can often take several days. As alternative methods, nucleic acid-based biosensors are of increasing importance as the need for faster, simpler, and cheaper methods for obtaining sequence-specific information grows.2 A number of biosensor approaches have been suggested in the recent past for the identification and quantification of DNA and RNA molecules. To obtain low limits of detection, they can be combined with the polymerase chain reaction (PCR) or with other replication systems, such as the reverse-transcriptase PCR (RT-PCR), the ligase chain reaction (LCR), the self-sustained sequence reaction (3SR), and nucleic acid sequence based amplification (NASBA).3 NASBA is generally a better-suited amplification system for the detection of viable organisms because of its isothermal nature and the fact that it exclusively amplifies RNA.4 Biosensors based on optical detection with a variety of different principles have been suggested, such as those based on intercalating dyes,5 molecular beacons,6,7 simple visual or reflectance (1) Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry; Worth Publishers: New York, 2000; pp 1132-1133. (2) Wang, J. Nucleic Acids Res.. 2000, 28 (16), 3011-3016. (3) Iqbal, S. S.; Mayo, M. W.; Bruno, J. G.; Bronk, B. V.; Batt, C. A.; Chambers, J. P. Biosens. Bioelectron. 2000, 15, 549-578. (4) Baeumner, A. J.; Humiston, M. C.; Montagna, R. A.; Durst, R. A. Anal. Chem. 2001, 73, 1176-1180. (5) Junhui, Z.; Hong, C.; Ruifu, Y. Biotechnol. Adv. 1997, 15 (1), 43-58. (6) Bernacchi, S.; Mely, Y. Nucleic Acids Res. 2001, 29 (13), e62. 10.1021/ac034945l CCC: $27.50

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detection,8,9 or microfluidic biosensors using dye-entrapping liposomes10,11 reaching low detection limits in the low picomolar range. Electrochemical biosensors have been suggested that use carbon electrodes with horseradish peroxidase as signal amplification system12 or methylene blue as intercalating dye13 and enzyme amplification systems14 achieving low femtomolar detection limits. Other transduction principles include microcantilevers,15 the quartz crystal microbalance,16 and surface plasmon resonance.17 Although DNA microarrays represented a revolution in genomic sequence analysis just a decade ago, they are now routinely used for genome sequencing,18 mRNA expression monitoring,19 mutation analysis,20-22 detection of disease markers,23 and drug discovery.24 Costs of fabrication are still high; nonetheless, some companies provide their customers with custom-made arrays for independent experiments. Although DNA microarrays do not fall into the category of simple to use, inexpensive, and portable biosensor, they do permit high-throughput analysis of entire genomes in one experiment and utilize microfabrication techniques for the site-selective immobilization of their biorecognition elements. All of the biosensors currently available for the specific detection of target DNA and RNA sequences require a series of experiments in order to be converted to the detection of a different target sequence. Aside from finding new detection probe sequences, the probes typically have to be labeled in a specific manner, which in most cases is a lengthy procedure to be carried out by well-trained technicians. In addition, the probes have to be purified, stabilized, and tested and the assay conditions reoptimized for the newly labeled probes. Therefore, it is currently (7) Liu, X.; Farmerie, W.; Schuster, S.; Tan, W. Anal. Biochem. 2000, 283, 5663. (8) Hartley, H.; Baeumner, A. J. Anal. Bioanal. Chem. 2003, 376 (3), 319327. (9) Baeumner, A. J.; Schlesinger, N. A.; Slutzki, N. S.; Romano, J.; Lee, E. M.; Montagna, R. A. Anal. Chem. 2002, 74, 1442-1448. (10) Kwakye, S.; Baeumner, A. J. Anal. Bioanal. Chem. 2003, 376 (7), 10621068. (11) Esch, M. B.; Locascio, L. E.; Tarlov, M. J.; Durst, R. A. Anal. Chem. 2001, 73, 2952-2958. (12) Campbell, C. N.; Gal, D.; Cristler N.; Banditrat, C.; Heller, A. Anal. Chem. 2002, 74, 158-162. (13) Meric, B.; Derman, K.; Ozkan, D.; Kara, P.; Erensoy, S.; Akarca, U. S.; Mascini, M.; Ozsoz, M. Talanta 2002, 56, 873-846. (14) Zhang, Y.; Kim, H.-H.; Heller, A. Anal. Chem. 2003, 75, 3267-3269. (15) Fritz, J.; Baller, M. K.; Lang, H. P.; Rothuizen, H.; Vettiger, P.; Meyer, E.; Guntherodt, H. J.; Gerber, C.; Gimzewski, J. K. Science 2000, 288 (5464), 316-318. (16) Mo, X. T.; Zhou, Y. P.; Lei, H.; Deng, L. Enzyme Microb. Technol. 2002, 30 (5), 583-589. (17) Feriotto, G.; Borgatti, M.; Mischiati, C.; Bianchi, N.; Gambari, R. J. Agric. Food Chem. 2002, 50 (5), 955-962. (18) Chee, M.; Yang, R.; Hubbell, E.; Berno, A.; Huang, X. C.; Stern, D.; Winkler, J.; Lockhart, D. J.; Morris, M. S.; Fodor, S. P. A. Science 1996, 274, 610614. (19) Lockhart, D.; Dong, H.; Byrne, M.; Follettie, M.; Gallo, M.; Chee, M.; Mittmann, M.; Wang, C.; Kobayashi, M.; Horton, H.; Brown, E. Nat. Biotechnol. 1996, 14 (13), 1675-1680. (20) Kozal, M. J.; Shah, N.; Shen, N.; Yang, R.; Fucini, R.; Merigan, R.; Richman, D.; Morris, D.; Hubbell, E.; Chee, M. S.; Gingeras, T. G. Nat. Med. 1996, 2 (7), 753-759. (21) Sosnowski, R. G.; Tu, E.; Butler, W. F.; O’Connell, J. P.; Heller, M. J. PNAS 1997, 94, 1119-1123. (22) Gerry, N. P.; Witowski, N. E.; Day, J.; Hammer, R. P.; Barany, G.; Barany, F. J. Mol. Biol. 1999, 292, 251-262. (23) Witowski, N. E.; Leiendecker-Foster, C.; Gerry, N. P.; McGlennen, R. C.; Barany, G. Biotechniques 2000, 29, 936-939. (24) Debouck, C.; Goodfellow, P. N. Nat. Genet. 1999, 21, 48-50.

not possible to replace the lengthy aforementioned blotting procedures with a simple biosensor for rapid nucleic acid analysis in the laboratory. Although instruments such as the LightCycler ameliorate this situation for some applications, high costs often prevent their use in many laboratories. We therefore suggest a simple universal simple biosensor that can be made specific for the detection of any DNA or RNA sequence by a straightforward 10-min incubation at 41 °C. In addition, generally no optimization of assay conditions is required. The universal biosensor is based on earlier, specific biosensors developed in our laboratory.8,9,25 It utilizes two universal components: (1) dye-entrapping liposomal nanovesicles that bear generic oligonucleotides on their outer surface and (2) polyethersulfone membranes with streptavidin immobilized in the detection zone. The two specific elements are the reporter and capture probes. The reporter probes consist of two segments: the 3′ end is complementary to the generic oligonucleotide bound to the nanovesicle, and the 5′ end is complementary to the target sequence. The capture probes are purchased already biotinylated at their 5′ end and are complementary to a different portion of the target sequence. By mixing and incubating the liposomes, reporter probes, target sequence, and capture probes with a hybridization buffer for 20 min at 41 °C, a sandwich is formed. The mixture is pipetted onto the polyethersulfone membrane and allowed to migrate along the strip via capillary action for ∼8 min. If the target sequence is present, the complex will be captured in the detection zone via streptavidin-biotin binding. Thus, the amount of liposomes present in the detection zone is directly proportional to the concentration of target sequence in the sample. Including signal quantification using a portable reflectometer (or visual detection), the assay can be completed within 30 min. The general principle of the biosensor is shown in Figure 1. This universal biosensor should find its application in molecular and microbiological research laboratories as substitution to the lengthy Northern and Southern Blot analyses and to expensive instruments, such as the LightCycler, help in the development of specific biosensor assays by rapidly screening detection sequences of interest as well as any testing situation in which nanomolar concentrations of nucleic acid sequences need to be rapidly identified and quantified. MATERIALS AND METHODS Reagents. All general chemicals and buffer reagents (reagent grade or above) were obtained from Sigma Company, St. Louis, MO. Organic solvents were purchased from Aldrich Chemical Co., Milwaukee, WI. Predator membranes were obtained from Pall/ Gelman Company, Port Washington, NY. Lipids were purchased from Avanti Polar Lipids, Alabaster, AL. Sulforhodamine B and streptavidin were acquired from Molecular Probes Company, Eugene, OR. All nucleic acid sequences, probes, primers, and synthetic targets with the appropriate modifications, were purchased from Qiagen, Valencia, CA. Sequences. In Table 1, all of the sequences used in this study are summarized. The melting temperatures of the capture probes and of the specific portions of the reporter probes calculated at 50 mM salt concentration are as follows: Escherichia coli RP (55 °C), CP (52.1 °C); Bacillus anthracis RP (53.1 °C), CP (50.4 °C); and Cryptosporidium parvum RP (49.2 °C), and CP (62 °C). (25) Baeumner, A. J.; Cohen, R. N.; Miksic, V.; Min, J. H. Biosens. Bioelectron. 2003, 8 (4), 405-419.

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Figure 1. The set-up for a universal biosensor assay. The universal liposomal nanovesicles bear dC-rich oligonucleotides covalently attached to their surface. The universal polyethersulfone membranes have streptavidin immobilized in the detection zone. During a biosensor assay, the nanovesicles are mixed with a reporter probe that has a dG-rich sequence in tandem with a target-specific sequence, the target DNA or RNA sequence, and a biotinylated capture probe also specific for the target DNA/RNA. This hybridization mixture is incubated in a test tube for 20 min at 41 °C. Subsequently, the membrane is inserted into the tube so that the mixture migrates up the strip based on capillary action. After ∼8 min, the amount of liposomes can be quantified in the detection zone and correlated directly to the concentration of target sequence in the sample. Table 1. DNA Sequences of Oligonucleotides Used in the Development and Optimization of the Universal Biosensor namea

DNA sequence (5′-3′ orientation)b

generic probe

CCA CCC CCA CCC CCA CCC CC

E. coli reporter probe capture probe clpB STS

GTC TGG TGA ATT GGT TCC GGG GGG TGG GGG TGG GGG TGG CCG TTG GCA CAG CAA ATA GGC AAC CGT GTC GTT TAT CAG ACC ACT TAA CCA AGG C

B. anthracis reporter probe capture probe AtxA STS

CAA GAT GTC CGC GTA TTT ATG GGG GTG GGG GTG GGG GTG G CTA GAA ATA TCG GGA AGA GAA AT AAA TAC GCG GAC ATC TTG TC TTC TCT TCC CGA TAT TTC Tag

C. parvum reporter probe capture probe hsp70 STS

GTG CAA CTT TAG CTC CAG TTG GGG GTG GGG GTG GGG GTG G AGA TTC GAA GAA CTC TGC GC A CCA GCA TCC TTG AGC ATT TTC TCA ACT GGA GCT AAA GTT GCA CGG AAG TAA TCA GCG CAG AGT TCT TCG AAT CTA GCT CTA CTG ATG GCA ACT GAA

b STS indicates the synthetic target sequence used for detection. The capture probes are biotinylated at the 5′ end. The generic probe is modified with an amine group at the 5′ end. b The portions of the reporter probes that bind to the target sequences are underlined.

Preparation of Membranes. Polyethersulfone membranes were cut into 7.5-cm by 4.5-mm strips. Streptavidin was diluted in 0.4 M Na2CO3/NaHCO3 buffer, pH 9.0, containing 5% methanol so that its final concentration was as desired (typically 20 pmol/ µL). One microliter of the prepared mixture was deposited onto the designated capture zone of each membrane strip exactly 2.5 cm from the bottom of the strip. Each spot was ∼3-4 mm long and covered the width of the strip. The membranes were then dried, first under a fume hood for 5 min, then in a vacuum oven at 53 °C and 15 psi, for 1.5 h. The membranes were then blocked by soaking them in a blocking reagent (0.5% poly(vinylpyrrolidone) (PVP) and 0.015% casein in Tris buffered saline (0.02 M Tris base, 0.15 M NaCl, 0.01% NaN3, pH 7.0)) for 30 min on a shaker at room temperature and blotted dry with tissue paper. They were allowed to fully dry in a vacuum oven at 25 °C, 15 psi, for 2-3 h and, finally, stored until use in vacuum-sealed bags at 4 °C. Preparation of Liposomal Nanovesicles. Liposomal nanovesicles were prepared using a slightly modified protocol of the reversed-phase evaporation method.9 Initially, 7.2 µmol (5.0 mg) 890 Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

dipalmitoyl phosphatidylethanolamine (DPPE) was dissolved in 1 mL of 0.7% triethylamine in chloroform by 1 min sonication. Subsequently, 14.3 µmol (3.5 mg) N-succinimidyl-S-acetylthioacetate (SATA) was added. The mixture was sonicated again and incubated for 20 min, forming a DPPE-ATA compound. To remove the triethylamine, 3 mL of chloroform was added to the mixture and evaporated under vacuum in a rotary evaporator at 45 °C. Finally, 1 mL of chloroform was added to this product. Subsequently, the DPPE-ATA, 40.3 µmol (0.0296 g) of dipalmitoyl phosphatidylcholine (DPPC), 21.0 µmol (0.015 g) of dipalmitoyl phosphatidylglycerol (DPPG), and 51.7 µmol (0.020 g) of cholesterol were dissolved in a mixture of chloroform, methanol, and isopropyl ether in a 6:1:6 ratio by sonication in a round-bottom flask in a water bath at 45 °C. To the lipid mixture, a total of 4 mL of a 150 mM sulforhodamine B (SRB) in 0.02 M phosphate buffer (K2HPO4/KH2PO4), pH 7.5 (516 mmol/kg), was added and sonicated for 5 min. The organic solvents were evaporated in a rotary evaporator so that liposomes formed, spontaneously entrapping SRB. To obtain a uniform particle size, the liposomes

subsequently were extruded through 2-µm, then 0.4-µm, filters (each 11 times) using the Avanti miniextruder and polycarbonate filters from Avanti Polar Lipids, Alabaster, AL. Liposomes were purified from free dye by gel filtration using a Sephadex G50 column and a 0.01 M PBS buffer, pH 7.0, containing sucrose to increase the osmolarity to 590 mmol/kg, followed by dialysis against the same buffer. Coupling of dC Probe to Liposome Surface. To couple the generic oligonucleotide to the outside of the liposomal nanovesicles, the probe was modified with an amino group at its 5′ end and dissolved in 0.05 M potassium phosphate buffer containing 1 mM EDTA, pH 7.8, to a concentration of 300 nmol/mL. A solution of N-(κ-maleimidoundecanoyloxy)sulfosuccinimide ester (sulfoKMUS) dissolved in dimethyl sufloxide (DMSO) was prepared at 10 mg/mL (20.8 µmol/mL), and this stock was added to the dissolved probe at a molar ratio of 3:1. Thus, for 30 nmol of dissolved probe, 4.3 µL of a 10 mg/mL sulfo-KMUS in DMSO solution was added. This mixture was incubated for 2-3 h to derivatize the amino-modified nucleotide probes with maleimide groups. The ATA groups on the liposomes were deprotected by deacetylating the acetylthioacetate groups on the surface of the liposomal nanovesicles, generating sulfhydryl groups. Hydroxylamine hydrochloride (0.5 M) in 0.4 M phosphate buffer pH 7.5 (K2HPO4/KH2PO4) with 25 mM EDTA was added to the volume of liposomes at a ratio of 0.1 mL hydroxylamine solution per 1 mL liposome solution, such that the final concentration of hydroxylamine was 0.05 M. The deacetylation reaction was allowed to proceed in the dark, at room temperature, and on a shaker for 2 h. At the end of both incubation periods, the pH of both mixtures was adjusted to 7.0 using 0.5 M KH2PO4. Then, for actual conjugation of the probe to the liposomes, the SH-tagged liposomes and the maleimide-derivatized nucleotide probe were mixed and allowed to react on a shaker at room temperature for 4 h, and then overnight at 4 °C. To quench the excess SH groups on the liposomes and the unreacted sulfo-succinimidyl groups on the sulfo-KMUS, ethylmaleimide and Tris were added equivalent to 10 times the molar quantity of SH and 20 times the molar quantity of sulfo-KMUS, respectively. A 200 mM ethylmaleimide solution in 0.05 M Tris-HCl, 0.15 M NaCl, 0.1 M sucrose, pH 7.0 was prepared. The appropriate volume (37.6 µL for a 30 nmol preparation) was added to the liposomes and incubated for 30 min on a shaker. The tagged liposomes were purified from free reporter probe by gel filtration using a Sepharose CL-4B column and subsequently by dialysis using a 0.01 M PBS buffer, pH 7.0, 591 mmol/kg (osmolarity adjusted with sucrose). Liposomes were stored in the dark at 4 °C. Biosensor Assay. The biosensor assay was a general dipsticktype assay. All optimization experiments were carried out with E. coli sequences. First, for the coupling of the specific reporter probes to the liposomal nanovesicles and the formation of the target-probe complex, 2 µL of liposomes (0.2 mol % surface tag), 1 µL of reporter probe (2 pmol), 1 µL of target sequence, 1 µL of capture probe (1 pmol), and 5 µL of master mix (20% formamide, 4× SSC, 0.4% Ficoll, 0.4 M sucrose) were combined in a glass tube. This hybridization mixture was incubated at 41 °C for 20 min. After incubation, a membrane strip (with 20 pmol of

Figure 2. Positioning of reflectometer readings. The top and bottom of the membrane are indicated to give orientation. The bottom of the membrane is the end that is inserted into the glass tube, where migration of the hybridization mix begins. (1) Capture zone, the position on the membrane where a signal will occur. (2) Location on the strip where the background measurement was taken.

streptavidin) was inserted into the glass tube, and the hybridization mixture was allowed to migrate up the strip. Subsequently, 35 µL of running buffer (40% formamide, 8× SSC, 0.2% Ficoll, 0.2 M sucrose) was added to the glass tube and allowed to traverse the entire length of the strip. After 8-10 min, all of the running buffer had run the length of the strip, and the signal at the capture zone was analyzed with the BR-10 reflectometer (from ESECO Company, Cushing, OK). The reflectometer measures the reflectance of light at a wavelength of 560 nm, which is close to the maximum absorbance of the sulforhodamine B that is encapsulated within the liposomes. Each experimental strip had two measurements taken with the reflectometer. One measured the intensity of the signal at the capture zone. The other measured the level of background noise just below the capture zone, ∼2 cm above the bottom of the strip. The two zones chosen for signal detection are shown in Figure 2. In addition, a negative control containing water instead of the target sequence was included in every experiment to account for any variation of the protocol during the optimization. The concentrations of each component in both the master mix and the running buffer, the percent tag of generic probe on the liposome, length of generic probe, and the amounts of reporter and capture probe per assay were varied for optimization purposes. In addition, the incubation temperature and the sequence of component additions were also optimized. RESULTS The most important design criterion of the development of the universal nucleic acid biosensor was the simplicity with which it could be transformed into a sequence-specific assay. The generic sequence approach was favored over antibody-antigen interactions using reporter probes labeled with an antigen, since this binding event would rely on buffer requirements other than the nucleic acid hybridization itself. In contrast, the biotin-streptavidin system is known to work well with a variety of buffers and was therefore chosen as second “simple” binding chemistry. In addition, the liposome membrane-based biosensor format was chosen for its ease of use, proven sensitivity of detecting nucleic acid sequences in the low nanomolar range, and its speed (i.e., the assay itself takes a total of only 20 min). Liposomal nanovesicles entrapping hundreds of thousands of dye molecules generate sufficient signal for low nucleic acid concentrations so that visual or quantitative reflectance measurements are feasible.25 Alternatively, the membranes could be scanned using a computer scanner and the gray scale intensity measured for quantification if no Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

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reflectometer is available.26 The generic liposome probe sequence was chosen to contain as many C (or G) as possible in order to ensure a strong binding to the reporter probe, even under high stringency conditions, and to have no hairpin or probe dimer structures that could prevent a binding to the reporter probe. Different lengths were initially tested, that is, 17-nt-, 20-nt-, 25-nt-, and 30-nt-long sequences, in order to determine the length that is optimal under the given biosensor conditions. The 20-nt sequence gave the best signal-to-noise ratios and was therefore chosen for all subsequent experiments. Optimization of the Universal Biosensor Assay. Initial tests investigated the best incubation protocol for coupling of reporter and capture probes to the liposomal nanovesicles and the membranes, respectively. To optimize the hybridization reaction, the order of addition of the various components was investigated: (1) separate incubation of liposomes and reporter probes; (2) separate incubation of reporter probes and target sequence; (3) separate incubation of liposomes, reporter probes, and target sequence; (4) dot blotting of the capture probe onto the detection zone; (5) addition of the capture probe to the hybridization mixture just prior to the insertion of the membrane strip; and (6) variation of incubation time and temperature for the different incubation steps. It was experimentally determined that no difference was found between sequential incubation steps and mixing of all components at once, that is, liposomes, reporter and capture probes, and target sequence. Additional studies on the required incubation time are described further below. Subsequently, the components of the master mix were optimized. The concentrations of formamide (0, 10, 20, 30, 40, and 50%), SSC (3, 4, 5, 6, 7, 8, 9, and 10×), reporter probe (0.5, 1, 1.5, 2, 2.5, 3, 5, and 10 pmol per assay), and capture probe (0.5, 1, 2, 3, 4, 5, 10, and 25 pmol per assay) were investigated. In addition, the concentration of the generic probe on the liposomes (0.1, 0.2, 0.4, and 0.6% (lipid mole fractions)) was also optimized. Each component was optimized separately and then chosen as a parameter for subsequent experiments. For example, data obtained for the optimization of the generic probe amount on the liposomes are shown in Figure 3. A clear maximum is at 0.2% of the lipid mol fraction. Similar experiments had been done for an E. coli-specific biosensor25 in which 0.4 mol % specific reporter probe tags were found to be optimal. With respect to the running buffer optimization, formamide (0, 10, 20, 30, 40, and 50%) and SSC (3, 4, 5, 6, 7, 8, 9, and 10×) concentrations were varied as well as the amount of running buffer (30, 35, and 50 µL) added to the assay. The latter assay parameters had no significant impact on the signals, since excess buffer simply remained in the bottom of the glass tube. The amount of streptavidin on the polyethersulfone membrane was also varied (10, 15, 20, 25, and 30 pmol). Finally, the time and sequence of incubation were studied using two different approaches. In the first approach, all components of the hybridization mixture (liposomes, reporter probe, target sequence, capture probe, and master mix) were simultaneously incubated together for varying amounts of time (2, 5, 10, 15, 20, and 25 min). In the second one, the liposomes, reporter probe, target sequence, and master mix were incubated together for varying amounts of time (2, 5, 10, (26) Rule, G. S.; Montagna, R. A.; Durst, R. A. Clin. Chem. 1996, 42 (8), 12061209.

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Figure 3. Optimization of the liposome generic probe tag. Results from assays that used liposomes containing 0.1, 0.2, 0.4, and 0.6 mol % tags to detect 500 fmol of synthetic E. coli target are shown. Reflectometer signals are plotted versus the liposome surface tag. Average background signals are also shown and are in all cases similar to or lower than the signals of the negative controls (12 ( 1.7). Each data point represents an average (1 standard deviation of five replicates. The liposomes with the 0.2 mol % surface tag proved to give the strongest signal. Table 2. Optimal Conditions for the Universal Biosensor Assay hybridization mixture components 2 µL of liposomes (0.2 mol % surface tag) 1 µL of reporter probe (2 pmol) 1 µL target sequence 1 µL of capture probe (1 pmol) 5 µL master mix (20% formamide, 4× SSC, 0.4% Ficoll, 0.4 M sucrose) incubation conditions combine all components of the hybridization mixture incubate for 20 min at 41 °C membrane 20 pmol of streptavidin immobilized at the detection zone running buffer volume: 35 µL to be added following absorption of hybridization mixture composition: 40% formamide, 8× SSC, 0.2% Ficoll, 0.2 M sucrose

15, 20, and 25 min). Then the capture probe was added, followed by another 20-min incubation. Although there was no significant difference between the overall maximum signal height of either approach, it was found that a minimum of 15 min total incubation time was required (i.e., in the first approach, the signals dropped off to 70% with 10 min, 30% with 5 min, and only 15% with 2 min of incubation). No difference in signal was obtained for longer incubations. In comparison, a 10-min incubation of liposomes and target sequence in the hybridization buffer is required in most of the specific biosensors developed in our lab. Since approach 1 (one incubation step only) was easier to perform and resulted in the same or slightly better signals than approach 2, it was chosen for future experiments. The overall optimized universal biosensor conditions are summarized in Table 2. Detecting Synthetic DNA Sequences from B. anthracis, C. parvum, and E. coli with the Universal Biosensor. Doseresponse curves were generated for three different sequences, atxA from B. anthracis, hsp70 from C. parvum, and clpB from E. coli. To be able to quantify the detection limit and the dynamic

Table 3. Summary of Upper and Lower Limits of Detection Obtained for the Universal Biosensor Assays Detecting E. coli, C. parvum, and B. anthracisa limit of detection, fmol

E. coli, specific ind;1universal C. parvum, specific ind;1universal B. anthracis, specific ind;1universal

Figure 4. Determination of the limit of detection for the universal detection of E. coli. A standard dose-response curve is presented with reflectometer signals plotted against analyte concentrations. Experiments were carried out using optimized biosensor conditions (Table 2). Data are presented in a log scale along the x axis; thus, the negative control (water instead of target sequence used in the assay) is indicated as 0.1 fmol target sequence.

range of the assays, synthetic DNA sequences were designed that mimicked the reporter and capture probe regions of the authentic sequences. In the case of E. coli and B. anthracis, short synthetic sequences were used; in the case of C. parvum, a longer, 97-ntlong segment consisted of most of the hsp70 sequence of interest. These synthetic sequences had been designed previously for organism-specific biosensor developed in our lab. Their sequences are given in Table 1. The E. coli target sequence was investigated ranging from 1 to 100 000 fmol per assay (Figure 4). Triplicate analyses of each concentration were done. An excellent detection limit of at least 1 fmol (1 nM) was determined, calculated by adding 3 times the standard deviation of the negative control to its average value, and correlating that to a concentration on the curve, (i.e., x0 ) 16.7 ( 0.58. x0 + 3× sd ) 18.4 < 22.7 ( 3.1 (signal of 1 fmol)). The upper limit of the dynamic range was 750 fmol. Above target concentrations of 750 fmol, a classical hook effect, typical for sandwich assays, was noted. Although a quantitative analysis is obviously possible, the relatively high standard deviations limit this. In the future, the membrane-making process will be improved by employing a spraying technique used in thin-layer chromatography for the immobilization of the capture probes, thus creating lines of capture zone instead of round spots. This will result in more uniform signals in the capture zones and, thus, a more reliable reading with the reflectometer. Similar studies were done with C. parvum and B. anthracis. The data for lower and upper limits of detection are summarized in Table 3. In addition, the data are compared to those of the specific biosensors published earlier. It can be seen that all three assays using the universal biosensor were very similar to the specific biosensors or slightly better. The discrepancy between the specific and the universal assay for C. parvum detection is due to the difference in assay format. Esch and colleagues used a competitive approach in comparison to the sandwich format of the universal biosensor, resulting in a higher value for both the lower and upper detection limit as expected. Detection of Authentic RNA Sequences from B. anthracis, C. parvum and E. coli. All prior experiments used a synthetic target sequence in order to optimize the universal

lower

upper

5 1 80 1 1.5 1

1000 750 2900 1000 100 750

a The data were compared to those published earlier generated by biosensors specific for the respective organisms.8,25,27

biosensor assay and to determine lower and upper limits of detection. In this set of experiments, mRNA sequences isolated from the target organisms were amplified using nucleic acid sequence-based amplification (NASBA) and analyzed with both the universal and the specific biosensors. Details about the amplification reactions, including amplification primer sequences, are given in the respective publications.8,25,27 The specific biosensor for C. parvum has been transformed into a sandwich assay (data not published), which is used here for comparative purposes. Analyses were done in triplicates. Comparing the universal and the specific biosensors, it was again observed that there were no difficulties in detecting amplified RNA sequences with the universal biosensor and that, in fact, the signals were very similar. The universal signals for E. coli were 93% of the specific signal; for B. anthracis, 102%; and for C. parvum, 112% (standard deviations are in all cases around 15% or less). Thus, in all cases, the universal biosensor approach can be utilized; no specific biosensor would have to be developed for the specific sequences. CONCLUSION A vertical, sandwich assay for the rapid universal detection of any nucleic acid sequence (such as for the identification of pathogenic organisms as chosen here as example) has been successfully developed. The universal biosensor can be transformed into a specific assay via a simple incubation step of ∼10 min. No special training and no specific equipment other than a water bath or heating block are required in order to perform the analysis. Overall, any nucleic acid sequence can be identified and quantified within 30 min (i.e., 20 min of preincubation, plus 8-10 min for the membrane assay, whereas the specific biosensors typically require a 10-min preincubation step), as long as reporter and capture probe sequences are known. Signals can be evaluated qualitatively by visual detection or can be quantified using a computer scanner or a portable reflectometer obtaining lower limits of detection of 1 nM (i.e., 1 fmol per assay). The analytespecific regions of the reporter and capture probes tested here ranged from 18 nt to 21 nt in length and had Tm values between 49.5 and 62 °C. It is expected that probes with similar data will perform as well in the universal assay as the sequences tested here and that small signal decreases can be expected with much (27) Esch M. B.; Baeumner A. J.; Durst R. A. Anal. Chem. 2001, 73, 31623167.

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shorter or much longer probes (i.e., much lower and higher melting temperatures). In that case, the formamide and SSC concentrations in master mix and running buffer could be adjusted accordingly if necessary. Thus, if the sequences of reporter and capture probes are chosen to be specific for the target sequence, the biosensor will be able to provide sensitive, specific, and rapid quantitative analyses of any DNA or RNA sequence of interest. This universal biosensor is therefore ideal for research labs substituting laborious methods, such as Northern and Southern

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Blotting, improving techniques in which only agarose gel electrophoresis is used for sequence identification, and also for the development of specific biosensors, since many sequences can easily be tested with the same biosensor assay during the testing stage. Received for review August 12, 2003. Accepted November 8, 2003. AC034945L