Sequential Injection Analysis System for the Sandwich Hybridization

Anal. Chem. 2006, 78, 1958-1966

Sequential Injection Analysis System for the Sandwich Hybridization-Based Detection of Nucleic Acids Katie A. Edwards and Antje J. Baeumner*

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

A sequential injection analysis lab-on-valve (SIA-LOV) system was developed for the specific detection of singlestranded nucleic acid sequences via sandwich hybridization of specific DNA probes to the target sequence. One DNA probe was tagged with fluorescein; the other was biotinylated and immobilized to streptavidin-coated porous beads. The system was optimized with respect to buffer composition, length of hybridization and wash steps, and volumes and concentrations of components used. On-bead oligonucleotide hybridization was studied using UV detection at 260 nm, while a final dose response curve was quantified using fluorescence detection. A dynamic range of 1-1000 pmol was obtained for a synthetic DNA sequence that was homologous to a segment in the B. anthracis atxA mRNA. A within-day variation of 7.2% and a day-to-day variation of 9.9% was observed. Each analysis was completed within 20 min. Subsequently, the system was applied to the detection of atxA mRNA expressed in a surrogate organism and amplified using NASBA. The SIA-LOV will find its application in routine laboratory-based analysis of specific singlestranded DNA/RNA sequences. Future improvements will include the integration of dye-encapsulating liposomes for signal enhancement used in lieu of the single fluorophorelabeled probe in order to lower the limit of detection. The rapid and accurate identification of bacteria is of crucial importance in medical diagnostics,1 national security,2,3 water quality,4 and food production settings.5-7 Culturing suspect samples followed by biochemical or serological identification has been considered to be the gold standard for bacterial detection but is a time-consuming and laborious approach.8 Enzyme-linked immunosorbant assays, fluorescence in situ hybridization, poly* Corresponding author. Tel.: +1-607-255-5433. Fax: +1-607-255-4080. Email: [email protected]. (1) Peters, R.; van Agtmael, M.; Danner, S.; Savelkoul, P.; VandenbrouckeGrauls, C. Lancet Infect. Dis. 2004, 4, 751-760. (2) Iqbal, S.; Mayo, M.; Bruno, J.; Bronk, B.; Batt, C.; Chambers, J. Biosens. Bioelectron. 2000, 15, 549-578. (3) Higgins, J.; Ibrahim, M.; Knauert, F.; Ludwig, G.; Kijek, T.; Ezzell, J.; Courtney, B.; Henchal, E. Ann. N. Y. Acad. Sci. 1999, 894, 130-148. (4) Ehlers, M.; Grabow, W.; Pavlov, D. Water Res. 2005, 39, 2253-2258. (5) Ellis, D.; Goodacre, R. Trends Food Sci. Technol. 2001, 12, 414-424. (6) de Boer, E.; Beumer, R. Int. J. Food Microbiol. 1999, 50, 119-130. (7) Li, Y.; Zhuang, S.; Mustapha, A. Meat Sci. 2005, 71, 402-406. (8) Malorny, B.; Tassios, P.; Rådstro ¨m, P.; Cook, N.; Wagner, M.; Hoorfar, J. Int. J. Food Microbiol. 2003, 83, 39-48.

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merase chain reaction (PCR) in combination with a variety of detection principles ranging from agarose gel electrophoresis to real-time analysis, and biosensors have been developed as alternative detection systems in order to overcome the disadvantages of lengthy culture-based approaches. Sequential injection analysis is a versatile method that has been employed for the detection of inorganic and organic food components,9,10 compounds of pharmaceutical interest,11,12 and environmental contaminants.13-15 It has been utilized to automate many standard wet chemistry techniques16,17 and offers the potential for automation of some aspects of other biological-based laboratory assays.18-20 Miniaturized introduction ports, such as a lab-on-valve (LOV), provide ample opportunity for sample manipulation and require only microliter volumes of reagents yielding minimal waste. Bead injection analysis (BIA) techniques based on the sequential injection analysis platform offer several advantages over sequential or flow injection techniques.21,22 BIA provides a fresh surface for each sample, which reduces the possibility of surface fouling and sample carryover and avoids time-consuming column regeneration between analyses. Another reported advantage is a lower limit of detection, since analytes can accumulate on the surface of the beads rather than being diluted in the surrounding buffer.22 The use of nucleic acid probes immobilized on renewable surfaces in BIA has been reported previously for nucleic acid (9) Pe´rez-Olmos, R.; Soto, J.; Za´rate, N.; Arau´jo, N.; Lima, J.; Saraiva, M. Food Chem. 2005, 90, 471-490. (10) Reis Lima, M.; Fernandes, S.; Rangel, A. J. Agric. Food Chem. 2004, 52, 6887-6890. (11) Solich, P.; Pola´sek, M.; Klimundova´, J.; Ruzicka, J. TrAC, Trends Anal. Chem. 2004, 23, 116-126. (12) Paula, C. A. G.; Pinto, M.; Lu´cia, M. F. S.; Saraiva, Joa˜o L. M. Santos; Lima, J. L. F. C. Anal. Chim. Acta 2005, 539, 173-179. (13) Echols, R. T.; Christensen, M. M.; Krisko, R. M.; Aldstadt, J. H. Anal. Chem. 1999, 71, 2739-2744. (14) Cerda`, V.; Cerda`, A.; Cladera, A.; Oms, M. T.; Mas, F.; Go´mez, E.; Bauza´, F.; Miro´, M.; Forteza, R.; Estela, J. M. TrAC, Trends Anal. Chem, 2001, 20, 407-418. (15) Hu, Y.; He, Y.; Qian, L.; Wang, L. Anal. Chim. Acta, 2005, 536, 251-257. (16) Economou, A. TrAC, Trends Anal. Chem, 2005, 24, 416-425. (17) Alerm, L.; Bartroli, J. Anal. Chem. 1996, 68, 1394-1400. (18) Thordarson, E.; Jonsson, J. A.; Emneus, J. Anal. Chem. 2000, 72, 52805284. (19) Pollema, C. H.; Ruzicka, J.; Christian, G. D.; Lernmark, A. Anal. Chem. 1992, 64, 1356-1361. (20) Baxter, P. J.; Christian, G. D. Acc. Chem. Res. 1996, 29, 515-521. (21) Barnett, N.; Lenehan, C.; Lewis, S. Sequential injection analysis: an alternative approach to process analytical chemistry, TrAC, Trends Anal. Chem. 1999, 18, 346-353. (22) Ruzicka, J.; Scampavia, L. Anal. Chem. 1999, 71, 257A-263A. 10.1021/ac051768a CCC: $33.50

© 2006 American Chemical Society Published on Web 02/01/2006

Table 1. Capture, Reporter Probes, and NASBA Primers with Their Respective Modifications sequence (5′-3′) reporter probe synthetic target sequence biotinylated capture probe complementary capture probe primer 1 primer 2

CAAgATgTCCgCgTATTTAT-fluorescein ATAAATACgCggACATCTTgTCTTCTCTTCCCgATATTTCTAg biotin-CTAgAAATATCgggAAgAgAA ATAAATACgCggACATCTTg-biotin AATTCTAATACgACTCACTATAgggAgaagggggAAACggCCAATAATCA TCAAATTTgCgAAgAACTTgTA

purification23,24 and analysis,25 yet remains an underexplored area of research.26 Chandler and colleagues have demonstrated the principle for DNA analysis. For example, they used a renewable microcolumn packed with a covalently attached 15-nucleotide sequence on 60-µm beads for the isolation and purification of Geobacter chapellii DNA from soil extracts prior to PCR amplification,24 the use of immobilized 14-nucleotide DNA and PNA probes to capture rRNA from various bacteria using flow cytometry as a detection method,27 and a rotating rod as a means to introduce and remove probe-labeled beads for nucleic acid analysis using complementary probe-labeled target.25 These authors also demonstrated that the sandwich-hybridization principle for the detection of PCR-amplified DNA was successful in their renewable media sequential injection analysis (SIA) system. The system described in this report expands on these principles by investigating the detection of nucleic acid sequencebased amplification (NASBA) mRNA and further demonstrates the utility of the sandwich hybridization SIA format. A beadimmobilized biotinylated DNA capture probe and a fluoresceinlabeled DNA reporter probe hybridize specifically to an unlabeled synthetic DNA target, which was complementary to both probes for method development. Using a LOV28 device, the beads were situated between fiber-optic lines connected to a spectrophotometer, which allowed for monitoring of hybridization of the sequentially introduced target and reporter probe on the surface of the beads. Upon formamide-induced dissociation of the sandwich complex, detection of the released reporter probe using a downstream detector measured fluorescent signal proportional to the amount of target nucleic acid originally present. Subsequently, the optimized system was applied to the detection of Bacillus anthracis atxA mRNA expressed in an Escherichia coli strain and amplified using NASBA in order to prove the potential of this method for laboratory-based routine sample analysis. NASBA uses the enzymes avian myeloblastosis virus reverse transcriptase (AMV-RT), RNaseH, and T7 DNA-dependent RNA polymerase in the presence of deoxyribonucleoside triphosphates and appropriate primers to amplify relatively few copies of target RNA into billions of antisense copies isothermally at 41 °C within 90 min.29 (23) Bruckner-Lea, C.; Tsukado, T.; Dockendorff, B.; Follansbee, J.; Kingsley, M.; Ocampo, C.; Stults, J.; Chandler, D. Anal. Chim. Acta 2002, 469, 129140. (24) Chandler, D.; Schuck, B.; Brockman, F.; Bruckner-Lea, C. Talanta 1999, 49, 969-983. (25) Bruckner-Lea, C.; Stottlemyre, M.; Holman, D.; Grate, J.; Brockman, F.; Chandler, D. Anal. Chem. 2000, 72, 4135-4141. (26) Chandler, D.; Brockman, F.; Holman, D.; Grate, J.; Bruckner-Lea, C. TrAC, Trends Anal. Chem. 2000, 19, 314-321. (27) Chandler, D.; Jarrell, A. Anal. Biochem. 2003, 312, 182-190. (28) Ruzicka, J. Analyst 2000, 125, 1053-1060. (29) Deiman, B.; van Aarle, O.; Sillekens, P. Mol. Biotechnol. 2002, 20, 163179.

The advantages of NASBA over other amplification techniques such as PCR or RT-PCR include its rapid and selective amplification of RNA, its isothermal conditions, and the generation of singlestranded RNA.30,31 The low temperature (41 °C) employed during NASBA is not sufficient to denature genomic dsDNA; thus, this material is not coamplified along with the target RNA even without DNAse treatment of the sample.30 This property is advantageous since the detection of short-lived RNA is necessary for the detection of viable bacteria, not pathogens that may have been neutralized through chemical disinfection of environmental matrixes or pharmaceutical intervention in human or veterinary medicine. MATERIALS AND METHODS Reagents. All reagents used were molecular biology grade, unless otherwise specified. The sequences of the synthetic DNA probes and primers for NASBA were purchased from Operon (Alamada, CA) and are listed in Table 1. The NASBA enzyme cocktail was purchased from Life Sciences, Inc. (St. Petersburg, FL), and the nucleotides used in the reaction were obtained from Bioline (Randolph, MA.) Sepharosestreptavidin beads were purchased from Zymed, Inc. (San Francisco, CA.) E. coli SG12036-pIu121, which expresses the atxA gene, was purchased from ATCC (Manassas, VA); sulforhodamine B and Oligreen reagent were purchased from Molecular Probes (Eugene, OR.) Instrumentation. The MicroSIA sequential injection analysis system was purchased from FiaLab Instruments (Seattle, WA) and was equipped with a syringe pump using a 500-µL syringe and a LOV multiport injector, which was interfaced to a PMT fluorescence detector (FiaLab Instruments) through port 2 of the LOV. A halogen/deuterium light source and miniature UV/visible spectrometer (both manufactured by Ocean Optics, Dunedin, FL) were connected through ports above port 2 in the LOV by 600µm-diameter fiber-optic cables (Figure 1A). The volume of the flow cell (∼2 µL; Figure 1B) for UV/visible detection was determined by the distance between the fiber-optic cables. The system was controlled by FiaLab for Windows software. Data analysis for the fluorescence aspect of the work was accomplished using an external software package for peak integration, Exachrom 3.0, which was purchased from Brechbu¨hler AG (Spring, TX.) Bead Preparation. The beads used for these analyses were composed of a streptavidin-Sepharose-4B conjugate. Sepharose 4B is a porous, non-cross-linked material containing 4% agarose with a mean bead diameter of 90 µm and distribution ranging from 45 to 165 µm. Its reported optimal molecular weight separation (30) Cook, N. J. Microbial Methods 2003, 53, 165-174. (31) Keer, J.; Birch, L. J. Microbiol. Methods 2003, 1764, 1-9.

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Figure 1. (A) Outline of the sequential injection lab-on-valve (LOV) system for DNA/RNA quantification using on-bead UV/visible and postbead fluorescence detection. All flow rate and volume manipulations are controlled through the syringe pump, which is situated between the inlet to the LOV and the hybridization buffer. The center groove rotates about a circular path (- - -) under software control to aspirate and dispense precise volumes of capture probe conjugated Sepharose 4B beads (port 6), synthetic DNA, or NASBA amplified RNA samples (port 5), fluoresceinlabeled reporter probe (port 4), and, last, formamide (port 3) through port 2. Unbound material/waste was thoroughly washed from the detection path before the next addition and removed either directly through port 1 or through the in-line valve after port 2. (B) The capture probe-conjugated beads are held in place between two fiber-optic cables connected to a halogen/deuterium light source and UV/visible spectrophotometer. The beads are prevented from passing through the tubing leading to the fluorescence detector by a 3-mm length of 0.0635-mm-i.d. PEEK tubing, which was held in place by 0.178-mm-i.d. tubing connecting the LOV to the fluorescence detector. (C) Fluorescence detection is accomplished by a PMT-based detector, which monitors the effluent from the beads. As the final analytical step to this sequence, formamide from port 3 in the LOV is used to rapidly dissociate the sandwich hybridization complex leading to a quantifiable peak. (D) On-bead detection is accomplished using the light source and detector connected through fiber-optic cables. This was used to monitor the extent of hybridizations after the addition of target sequence (260 nm) and reporter probe (489 nm). Assay control and data acquisition were carried out via software on the connected PC. Further method details are provided in the Materials and Methods section. The drawing is representative of the whole system, but elements are not to scale.

range is 70-20 000 kDa.32 The streptavidin/agarose ratio reported by the manufacturer was 1 mg of streptavidin/mL of Sepharose gel, which correlated to a binding capacity of 1.5-2.5 mg of biotinylated rabbit IgG/mL of streptavidin-conjugated gel.33 The 50% (w/v) Sepharose-streptavidin bead suspension was aliquotted into 1000-µL portions and centrifuged at 3000 rpm for 5 min to remove the supernatant. The volume was replaced with 1×PBS and 50 µL of the biotinylated capture probe (300 pmol/µL in 50 mM potassium phosphate buffer, pH 7.8 containing 1 mM EDTA) was added. The mixture was incubated at ambient temperature for 20 min and then was centrifuged and washed three times with 1×PBS to ensure that the amount of free capture probe present in the mixture was minimized. The DNA content of the supernatants was determined using the Oligreen reagent according to the manufacturer’s instructions. Oligreen is a fluorescent reagent that selectively binds to single-stranded DNA molecules and (32) Amersham Biosciences, Sepharose and Sepharose CL instruction sheet, 717098-00. (33) Zymed Laboratories, Sepharose-Streptavidin Conjugate, product specification sheet.

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permits their detection using fluorescence detection at λex ) 480 nm and λem ) 520 nm.34 The beads were then stored at 4 °C prior to use. Standard SIA Methodology. The 50% suspension of capture probe-conjugated beads (described in the previous section) was vortexed for 5 s and then added directly to port 6 (Figure 1A) of the LOV using a disposable microvolume transfer pipet. Since the beads settled within the port due to gravity, which hindered their direct aspiration, it was necessary to disperse them with buffer prior to each analysis. This was accomplished directly in the LOV by dispensing 10 µL of hybridization buffer (30% (v/v) formamide, 9×SSC (1.35 M sodium chloride, 0.135 M sodium citrate, 0.01% sodium azide, pH 7.0, 0.2% Ficoll type 400) to port 6 at 30 µL/s, and then 10 µL of the bead/hybridization buffer mixture was aspirated at 20 µL/s. The aspirated beads, which were then located in the center groove of the LOV, were introduced to the space between the fiber-optic cables above port 2 at a flow rate of 10 (34) Molecular Probes, MP07582, OliGreen ssDNA Quantitation Reagent and Kit, Product Information, January 13, 2001.

Table 2. Protocol of the SIA Method for the Detection of Single-Stranded Nucleic Acids step

LO port

volume (µL)

flow rate (µL/s)

syringe action

400

100

aspirate

6 6 2

10 10 200

30 20 10

dispense aspirate dispense

2 5 2

1 5

5 1

empty aspirate dispense

295 495 1 6

3 100 1 1

empty aspirate aspirate dispense

bead addition

target introduction

2 reporter probe introduction 4 2 2 signal generation 3 2

10 25 475

100 10 5 100 10 100 100

aspirate aspirate empty fill empty aspirate empty

300 50

100 100

aspirate aspirate

50 150

100 100 20

aspirate dispense empty

500 2 bead removal

500 1 2 2 1 2

empty

µL/s using a wash volume of 900 µL of hybridization buffer. The beads (total volume ∼2 µL) were held in place by a 3-mm-long PEEK tubing with an inner diameter of 0.0635 mm. This short piece of tubing was held in place by the 0.178-mm-i.d. tubing, which connected the LOV to the fluorescence detector. This setup allowed fluid to pass but retained the beads between the fiberoptic cables.35 One microliter of a sample consisting of either synthetic DNA or NASBA amplified RNA was aspirated from port 5 and directed over the beads using 5 µL of hybridization buffer at a flow rate of 1 µL/s, followed by a 5-min incubation period. Unbound materials were washed from the beads at 3 µL/s with 295 µL of hybridization buffer. This step was monitored at the spectrophotometer at 260 nm (Figure 1C). One microliter of fluorescein-labeled reporter probe (200 pmol/µL) was aspirated from port 4 and directed over the beads using 6 µL of hybridization buffer at a flow rate of 1 µL/s, followed by a 3-min incubation period. Unbound material was washed from the beads with 495 µL of running buffer at 10 µL/s using a software-controlled 24-V valve (Figure 1A) to direct wash to waste, rather than through the inline fluorescence detector. This step was monitored at the spectrophotometer at 489 nm (Figure 1B). Last, 25 µL of hybridization buffer followed by 475 µL of formamide was drawn through port 3 and dispensed over the hybridized complex at a rate of 5 µL/s, followed by 500 µL of hybridization buffer at 10 µL/s in order to generate the signal at the fluorescence detector, (35) Erxleben, H.; Manion, M.; Hockenberry, D.; Scampavia, L.; Ruzicka, J. Analyst 2004, 129, 205-212.

comment aspirate hybridization buffer begin loop (2×) disperse settled beads draw beads into center groove dispense beads over porous plug end loop wash residual capture probe from beads introduce sample into center groove center bolus of sample over beads incubate for 5 min remove unbound target from beads aspirate hybridization buffer introduce probe into center groove center bolus of probe over beads incubate for 3 min switch in-line valve to “on” remove unbound probe from beads switch in-line valve to “off” aspirate hybridization buffer fill syringe with formamide begin signal generation fill syringe with hybridization buffer complete signal generation aspirate hybridization buffer begin loop (2×) aspirate hybridization buffer remove beads from UV/visible flow cell 1-s delay remove beads from UV/visible flow cell discard used beads to waste fill syringe with hybridization buffer end loop

which was monitored using λex ) 485 nm and λem ) 530 nm filters. Bead removal was accomplished as follows: The syringe was filled with 500 µL of hybridization buffer and then emptied to waste. The following steps were repeated twice: The syringe was then filled with an additional 300 µL of hybridization buffer; 50 µL was aspirated from port 2 at 100 µL/s twice with a 1-s delay between aspirations; 150 µL was dispensed to waste (port 1) at 100 µL/s; and the remaining volume was dispensed through port 2 at 20 µL/s. The entire method, from bead injection to bead removal, was complete within 20 min. A summary of the sequential injection process is given in Table 2. To determine the optimal SIA protocol conditions with respect to the assay stringency, incubation times, reporter probe concentration, washing volumes, and the dissociation solution were optimized. The variations tested are summarized in Table 3. Only one parameter was changed at a time, and 100 pmol of synthetic target was used per analysis. Determination of Hybridization Efficiency. The on-bead hybridization efficiency was investigated using varying concentrations of synthetic target DNA (0, 1, 10, 50, 100, and 500 pmol/ µL) and the standard SIA methodology. The amount of unbound synthetic target DNA in the eluent was determined using the Oligreen reagent according to the manufacturer’s instructions. Similarly, the amount of unbound fluorescein-labeled reporter probe was determined after allowing the sandwich hybridization complex to be formed. Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

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Table 3. Optimization of the SIA Protocol. Conditions Investigated with Respect to Incubation Time, Probe Concentrations, and Dissociation Conditions optimization hybridization buffer incubation time probe concentration

dissociation conditions

parameter 1

parameter 2

0-50% formamide 0-10 min between target and capture probe fluorescein-labeled reporter probe (10-1000 pmol/µL)

0-20×SSC 0-10 min between reporter probe and hybridized target equal amounts of probe introduced through repetitive additions of lower probe concentration versus single introduction of high concentration

formamide deionized water 10 mM NaOH 30 mM OG 1% Triton X-100.

Bacteria Growth and Extraction. All bacterial strain sources and growth conditions were as described previously by Hartley.36 Briefly, E. coli SG12036-pIu121 was grown in 2% Lennox broth with 30 µg/mL tetracycline. Five to six colonies were inoculated into 5 mL of growth broth and grown for 4 h with vigorous shaking (200-250 rpm) at 37 °C. Then 250 µL of IPTG (50 mM) was added and the culture was grown for another 30 min to an optical density of ∼0.6 (at λ ) 600 nm). The E. coli SG12036-pIU121 cells were centrifuged, and the pellet was then treated with 100 µL of lysozyme (0.4 mg/mL) dissolved in 10 mM Tris/1 mM EDTA buffer, pH 8.0, to digest the cell wall. After a 5-10-min incubation period, the standard RNA extractions were carried out using a silica-guanidine thiocyanate protocol,37 provided as proprietary lysis buffer and isolation reagent modules in the RNeasy Total RNA kit (Qiagen). The standard bacterial protocol recommended by the manufacturer was followed. Total extracted RNA was quantified in a Beckman-Coulter spectrophotometer by measuring the absorbance at 260 nm (A260) and calculating the total yield based on one unit of absorbance at A260 corresponding to 40 µg of RNA/mL of water. The concentration of RNA was used to determine the appropriate dilution for the NASBA reaction, which was ∼0.01 µg/mL. The purity of the nucleic acid was measured by making a 1:100 dilution in 10 mM Tris-HCl buffered to pH 7.5 and analyzing the A260/A280 ratio. Nucleic Acid Sequence-Based Amplification. NASBA primers were mixed to a final concentration of 0.2 µM in 15% (w/v) dimethyl sulfoxide/1×NN buffer including 5 µL of the extracted RNA. NN buffer is composed of 40 mM ITP, 12 mM MgCl2, 70 mM KCl, 5 mM DTT, 1 mM each dNTP, 2 mM each NTP, 1.5 mM ITP, and 40 mM Tris. RNAse free water and previously determined positive RNA extract from E. coli SG120368-pIU121 (1 ng of RNA/5 µL, stored at -20 °C) were used as negative and positive controls, respectively. The mixture was heated at 65 °C for 5 min and then at 41 °C for 5 min. The three-enzyme cocktail (AMV-RT, RNase H, and T7 RNAP, 5 µL) was added and the reaction was incubated at 41 °C for 90 min. The success of the reaction was confirmed by lateral-flow assays developed by Hartley et al. (data not shown). (36) Hartley, H. A rapid and sensitive Bacillus anthracis biosensor. Masters Thesis, Cornell University, Ithaca, NY, 2002. (37) Boom, R.; Sol, C.; Salimans, M.; Jansen, C.; Wertheim van Dillen, P. J. Clin. Microbiol. 1990, 28, 495-503.

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RESULTS AND DISCUSSION The method presented in this work relied on the sequential injection of fluorophore-labeled probe and target sequence over an immobilized capture sequence to form a sandwich hybridization complex. The optimal conditions of the SIA system for the monitoring of hybridization efficiency were investigated by studying the influence of various parameters on its sensitivity and precision. Development and Optimization of the SIA-LOV Methodology. Biotinylated capture probes were immobilized to the porous streptavidin-conjugated Sepharose 4B beads to form the solid phase of the system. The amount of immobilized probe per microliter of bead solution was analyzed using Oligreen by quantifying the amount of capture probe left in the supernatant and wash solutions. A total of 72 ( 1.4% of added capture probe was retained with the beads yielding 60 ( 1.1 pmol of capture probe bound per microliter of streptavidin-Sepharose gel. Thus, ∼120 pmol of capture probe was present per assay for the 2-µL volume between the fiber-optic cables. The hybridization buffer was optimized with respect to stringency and volume used. While 300 µL of buffer was sufficient to remove unbound target sequences, 1695 µL of buffer was necessary to thoroughly flush unbound fluorescein-labeled reporter probe from the tubing connecting the bead seat to the fluorescence detector and thus avoid interference of the unbound reporter probe with the signal obtained through dissociation of the sandwich hybridization complex. This volume could be reduced to 495 µL when a software-controlled switch was placed directly after the bead seat, which sent unbound reporter probe to waste rather than to the fluorescence detector. The stringency of the hybridization buffer was adjusted using varying formamide and SSC concentrations with 100 pmol of synthetic target DNA and 100 pmol of reporter probe. The amount of hybridized target sequence remaining on the beads after washing with the hybridization buffer was quantified by the fluorescence peak area after the complex was released. In accordance with theory, as the amount of formamide in the hybridization mixture was increased, less hybridization occurred (Figure 2). The peak areas when 15 and 30% formamide were used along with 2×SSC were 95 and 41%, respectively, of that when no formamide was used in the presence of 2×SSC. Similar results were obtained when varying the SSC concentration between 0 and 20×. For example, when

Figure 2. Effect of varying the formamide composition of the mobile phase on the formation of a sandwich hybridization between capture probe, synthetic DNA target sequence, and the fluorescein-labeled reporter probe. The 100 pmol of synthetic target and 100 pmol of reporter probe were used in these experiments using a mobile phase composed of 2×SSC, 0.2% Ficoll, and 0-50% (v/v) formamide. Figure 3. Synthetic target addition onto capture probe-tagged beads. Detector was referenced with beads in the cell prior to addition. (A) 1 µL of synthetic DNA target solution was pushed onto bead surface within UV detector flow path. (B) After the hybridization period, unbound target was washed from detector. (C) Absorbance due to bound target remaining after washing.

the formamide concentration was held constant at 30% (v/v), 4×SSC was determined to yield the highest peak area. The signals at 3×SSC and 5×SSC were both ∼54% of the signal at 4×SSC, and no peak was observed above a concentration of 10×SSC. Combining the two stringency variations, conditions of 0% formamide and 4×SSC yielded the highest amount of sandwichhybridized complex leading to the maximum fluorescence signal. Previous research using the same sequences in a lateral-flow sandwich hybridization assay format yielded optimal hybridization conditions of 30% formamide and 9×SSC.38 Though 0% formamide yielded the highest peak area in this SIA system, the use of 30% formamide, as in the lateral-flow assays, was continued to ensure that the stringency would be sufficiently high to discriminate against nontarget organisms. Future work using nontarget organisms in the SIA system will determine whether this deviation from optimal conditions is necessary. For the optimization of the hybridization time, 1 µL of synthetic DNA (100 pmol) was introduced and the buffer flow was stopped to allow for varying contact times between the beads and the target sequence (0, 1, 3, 4, 5, 7, and 10 min). While the signal increased significantly between 0 and 5 min (1.8-fold) and a linear relationship between incubation time and signal was found, no additional hybridization occurred after 5-min incubation time. Additional experiments were carried out to study the hybridization of unlabeled target sequence to the immobilized capture probes using on-bead detection. These experiments avoided the additional complexity incurred through the second hybridization reaction forming a sandwich complex with the fluorescein-labeled reporter probe. The absorbance at 260 nm was monitored throughout the introduction and hybridization of DNA (∼6 min) and could be divided into three main stages: (a) the accumulation of target, (b) removal of unbound target from the bead surface using running buffer, and (c) leaving only bound target in the detection zone (Figure 3.) The amount of target sequence remaining bound to the bead surface after washing was found to be proportional to the amount injected (Figure 4.) The rise in absorbance at 260 nm levels off above 700 pmol of added synthetic target, which indicates that the upper limit that can be captured by the beads under the current hybridization

conditions has been reached. The hybridization efficiency was ∼78 ( 4.4% for introduced target amounts between 50 and 700 pmol. Similar results were obtained by Gingeras et al., who observed a constant percentage of hybridization (∼70%) even when a 100fold concentration range of complementary oligonucleotide target was used in their study of hybridization to a fixed concentration of dextran-immobilized probes.39 A substantially lower hybridization percentage below 50 pmol of introduced synthetic DNA target was observed and is likely due to the suboptimal heterogeneous hybridization conditions for low target amounts. A higher capture efficiency may possibly be attained if the contact time was extended well beyond 10 min, if the capture probe concentration on the beads was reduced, or if a blocking reagent on the beads was used to reduce repelling forces between probes and the bead surface. In addition, at the high immobilized probe concentrations employed in this study, steric hindrance may prevent all of the immobilized probe from being able to bind to target available in

(38) Hartley, H.; Baeumner, A. Anal. Bioanal. Chem. 2003, 376, 319-327.

(39) Gingeras, T.; Kwoh, D.; Davis, G. Nucleic Acids Res. 1987, 15, 5373-5390.

Figure 4. Amount of DNA target retained on the beads after the washing step (2×SSC, 30% formamide, 0.2% Ficoll). Target DNA was introduced over 2 µL of capture probe-immobilized beads and incubated for 5 min using the standard methodology. The absorbance values at 260 nm were taken at 400 s, i.e., after all unbound target had been washed from the beads. The experiment was done in a single analysis for each target concentration.


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solution. In fact, reducing the capture probe coverage may reduce the optimal time needed for hybridization. Peterson et al. reported on the hybridization kinetics of complementary and partially mismatched sequences with immobilized probe of high and low density.40 Complementary sequences at high concentration and ionic strength (1 µM target and 1 M NaCl, respectively) required several hours to reach steady-state hybridization when the immobilized probe concentration was high (3 × 1012 probes/cm2) yet only 1 h when the probe concentration was moderate (1.5 × 1012 probes/cm2). In addition, the efficiency of hybridization in the former case was only 50% at steady state versus 100% in the latter case. Yu et al. compared the hybridization efficiencies (HE) of a 15-mer target using a 15-mer streptavidin-immobilized biotinylated oligonucleotide probe and reported a HE of 96% when the probe density was 1.2 × 1012 molecules/cm2 but decreased with increasing probe density41 (84% HE when the probe concentration was 2.9 × 1012 molecules/cm2). The optimal amount of fluorophore-tagged reporter probe for the detection of bound target sequence was investigated by using 100 pmol of target DNA and varying concentrations of reporter probes (10-1000 pmol/µL). The amount of reporter probe remaining hybridized in the sandwich complex was assessed both by absorbance at 489 nm (data not shown, but trace similar to that shown in Figure 3) and by the fluorescence peak observed upon dissociation of the complex (Figure 2). Similar to what was observed before with the DNA target hybridization, only a fraction of the introduced fluorescein-labeled reporter probe remained bound to the target following its incubation over the bead surface. Since 100 pmol of synthetic target DNA was used, the amount of reporter probe ranged between 0.1× and 10× the amount of the added synthetic DNA. Thus, despite the large excess of reporter probe present during the hybridization time when high concentrations were introduced, the bulk of the probe was washed away leaving only a fraction of it bound, which is proportional to the original injected concentration. An excellent linearity between the resulting fluorescence peak area and the concentration of injected reporter probe was observed up to a concentration of 200 pmol of fluorescein-labeled reporter probe (Figure 5), leveling off for higher concentrations with worsened reproducibility (data not shown). Thus, the optimal reporter probe concentration was determined to be 200 pmol introduced into the system in 1 µL of solution. The increase in signal with increasing reporter probe concentration may be a function of the higher hybridization rate experienced with increasing probe concentration.42 Since the received analytical signal was proportional to the amount of reporter probe bound and also the amount of target retained on the beads, investigations were carried out to maximize the amount bound for both of these sequences. The effect of the reporter probe incubation time (0-10 min) was investigated, but a strong correlation between hybridization time and peak area was not observed. A contact time of 3 min was used for all future experiments. In the small volumes used, the hybridization kinetics are rapid enough that additional binding time, beyond the 5-min (40) Peterson, A.; Wolf, L.; Georgiadis, R. J. Am. Chem. Soc. 2002, 124, 1460114607. (41) Yu, F.; Yao, D.; Knoll, W. Nucleic Acids Res. 2004, 32, e75. (42) Keller, G.; Manak, M. DNA Probes, 2nd ed.; Macmillan Publishers Ltd.: New York, 1993; pp 1-26,


Figure 5. Investigation of varying fluorescein-labeled reporter probe concentrations by quantifying the peak area observed at the fluorescence detector. 100 pmol of synthetic DNA target and 10-200 pmol of fluorescein-labeled reporter probe were used per sample under otherwise standard methodology. Each point is the average of three peak area determinations; error bars represent one standard deviation. Table 4. Effect of the Number of Passes of Reporter Probe on the Degree of Hybridization

assay

[reporter probe] (pmol)

no. of passes

total [reporter probe] (pmol)

area

conc (%)a

A B C D

100 200 100 300

2 1 3 1

200 200 300 300

4 165 000 5 335 000 3 939 000 5 016 000

78 100 79 100

a This column shows the area of the multiple pass trial as a percentage of the area of the single pass trial using the same total picomoles of reporter probe

target hybridization and 3-min reporter probe hybridization, was not advantageous. Similar results were found by Chandler et al.; that is, no improvement was observed in the extent of hybridization when a contact time of 19 min was used versus a 3.5-min contact time.24 To better understand the hybridization that is occurring, the effect of using a high concentration of reporter probe (200 or 300 pmol) once versus using a lower concentration of probe (100 pmol) added two or three times, respectively, over the beads was investigated. The total amount of probe introduced was the same in both sets of experiments. In the latter case, the unbound reporter probe was thoroughly flushed from the beads prior to the next introduction (Table 4). It was found that high concentrations of reporter probe (B, D) yielded a higher degree of hybridization than using the equivalent concentration of reporter probe introduced through multiple passes (A and C, with 78 and 79%, respectively). This suggests that the higher rate of hybridization obtained with a higher concentration of reporter probe through mass action is more important than any competition effects that are avoided through multiple introductions. Similar results were found by Chandler et al. investigating the purification of DNA from soil samples. The effects of a single pass, versus multiple passes, of the DNA target over beads linked with a complementary sequence was investigated, and no improvement in capture efficiency between the two methods was observed.24

Figure 6. Dissociation of sandwich hybridization complex from beads using formamide, water, and 50 mM NaOH. The standard methodology was used with the substitution of water or 50 mM NaOH for the formamide used to wash the beads. Signals were recorded at the fluorescence detector.

The final SIA-LOV method was designed for fluorescence detection in order to obtain a low limit of detection, rather than using absorbance at 260 or 489 nm, which was used for the method development. Thus, the effect of the dissociation solution on the fluorescence signal was determined. Dissociation solutions investigated included formamide, deionized water, 50 mM NaOH, 30 mM n-octyl β-D-glucopyranoside (OG), and 1% Triton X-100 (Table 3). Each of these dissociation conditions was tried independently in an effort to generate a reproducible and quantitative fluorescence signal. Representative response curves observed at the fluorescence detector resulting from the dissociation of the hybridization sandwich for water, 50 mM NaOH, and formamide are shown in Figure 6. It was found that the rate of dissociation with reagents other than formamide was too slow; that is, long elution peaks were measured indicating that the dissociation was still not complete after 5 min. Similarly, while deionized water has been successfully used to dissociate hybridized DNA in other systems,24 experiments indicated that water-mediated dissociation did not yield a well-defined peak that was useful for quantification. In contrast, pure formamide accomplished this rapidly, generating a reproducible peak that directly correlated to the target concentration at the fluorescence detector within 2 min (Figure 7). Analytical Performance of the Sandwich-Hybridization SIA System. A dose response curve for the optimized SIA-LOV system was obtained using synthetic target DNA concentrations between 0.1 and 2000 pmol. The dynamic range was determined to be 3 orders of magnitude (1-1000 pmol) with a limit of detection at 1 pmol (Figure 8). The limit of detection was defined as the average height of the blank signal (3 replicates) + 3 StDev. The reproducibility of the assay was determined by analyzing a sample of synthetic target DNA (100 pmol) using the standard protocol, 200 pmol of reporter probe, and the same batch of capture probe-tagged beads on three separate days. The peak height over all analyses was 131 182 ( 12 948 (n ) 11). A withinday variation of 7.2% and a day-to-day variation of 9.9% was observed. The injection-to-injection variability likely stems from slight differences in the quantity of capture probe-tagged beads

Figure 7. Fluorescence signal obtained upon dissociating sandwich hybridization complexes from the beads using formamide. Standard methodology was used with varying DNA target concentrations. The peak height was used for quantitative measurements.

available for each analysis and the range of bead sizes present for this material. Detection of atxA mRNA Amplified Using NASBA. RNA samples from E. coli expressing atxA were extracted, amplified with NASBA, and then detected in the SIA-LOV system (Figure 9) in order to show that the method could be used to detect RNA extracted from bacteria and is not limited to synthetic DNA sequences. The samples were also analyzed using lateral-flow assays in order to prove that the extraction and NASBA reaction were successful (data not shown). Good qualitative correlation of the SIA and the lateral-flow tests was found; that is, all positive lateral-flow assays were also positive using SIA, and all negative samples were also identified correctly to be negative using SIA. Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

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mined here, a full NASBA amplification to the level of a few picomoles of RNA is required for successful detection. Thus, if only a low number of cells and thus limited numbers of target RNA sequence are present in the sample, a 90-min NASBA reaction is required, which drops to less than 60 min of amplification if more RNA is present.36

Figure 8. Dose response curve for the synthetic DNA target sequence (0.1-2000 pmol) using fluorescence detection and an optimized SIA-LOV protocol. Analyses were done in triplicates.

Figure 9. Representative fluorescence response curves of a positive (upper curve) and negative (lower curve) NASBA reaction. For the positive reaction, atxA mRNA was amplified; for the negative reaction, water was used instead. The SIA analysis was carried out using standard methodology.

NASBA is a powerful technique for RNA amplification but generally requires an external method for analysis such as gel electrophoresis and staining by ethidium bromide. One advantage to the SIA method, and other techniques based on sandwich hybridization, for amplified nucleic acid detection is the inherent degree of additional specificity due to hybridization of the amplicon between two target-specific probes. While different organisms were not studied for the development of this method, previous work in our laboratory has shown a high degree of specificity for the probes used here in the detection of B. anthracis versus other closely related organisms.36 Based on the detection limit deter-


CONCLUSIONS A sequential analysis system based on the sandwich hybridization of oligonucleotide sequences was developed. The entire assay time, from bead injection to bead removal, was 20 min. The method was capable of detecting 1-1000 pmol/µL of sample containing synthetic DNA and also was shown to detect RNA amplified by NASBA from bacteria samples. While the limit of detection is ∼3 orders of magnitude higher than the one determined for the liposome-based lateral-flow assay used as comparison technology in this paper, it proves to be a rugged and useful laboratory-based technology for higher throughput analyses with the capability of autosampling similar to other SIA methods. However, better sensitivity could be attained through the use of fluorescent dye encapsulating liposomes instead of the single labeled-fluorophore reporter probe. The release of hundreds of thousands to millions of dye molecules, coupled with fluorescence detection, should generate a limit of detection for the SIA system that would at least match the detection limit of the lateral flow assays (∼1 fmol/µL). In addition, it was found that only a fraction of the target that is introduced actually binds to the immobilized capture probe. That the hybridization of target sequences to bead-immobilized probes is subject to probe length and density effects,40 as well as surface charge and chemistry,26 has also been reported by others. Thus, improvement of the current SIA-LOV assay could be made with the goal of retaining more target sequence on the beads through increasing the spacer length between the capture probe and its biotin linkage to enhance conformational flexibility, decreasing the density of capture probe on the beads in the event that the high concentration adversely affects the hybridization kinetics,40 and investigating other porous and nonporous stationary phases. ACKNOWLEDGMENT The authors are grateful for the SIA training provided by Dr. Holger Erxleben and Dr. Jaromir Ruzicka at the University of Washington. Also, we thank Barbara Leonard for growing and extracting the bacterial strains used in this study.

Received for review October 3, 2005. Accepted December 27, 2005. AC051768A

Sequential Injection Analysis System for the Sandwich Hybridization

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