Isothermal DNA Amplification Coupled with DNA ... - ACS Publications

Nov 11, 2005 - Harvey Mudd College, Claremont, California 91711. We present a simple, rapid method for detecting short. DNA sequences that combines a ...
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Anal. Chem. 2005, 77, 7984-7992

Isothermal DNA Amplification Coupled with DNA Nanosphere-Based Colorimetric Detection Eric Tan,† Jennifer Wong,† Doris Nguyen,† Yolanda Zhang,† Barbara Erwin,† Lori K. Van Ness,† Shenda M. Baker,‡ David J. Galas,† and Angelika Niemz*,†

Keck Graduate Institute of Applied Life Sciences, 535 Watson Drive, Claremont, California 91711, and Harvey Mudd College, Claremont, California 91711

We present a simple, rapid method for detecting short DNA sequences that combines a novel isothermal amplification method (EXPAR) with visual, colorimetric readout based on aggregation of DNA-functionalized gold nanospheres. The reaction is initiated by a trigger oligonucleotide, synthetic in nature for this proof-of-principle study, which is exponentially amplified at 55 °C and converted to a universal reporter oligonucleotide capable of bridging two sets of DNA-functionalized gold nanospheres. This reaction provides >106-fold amplification/conversion in under 5 min. When combined with a solution containing DNA nanospheres, the bridging reporter causes nanosphere aggregation. The resulting color change from red to dark purple or blue is enhanced through spotting the solution onto a C18 reversed-phase thin-layer chromatography plate. The reaction can easily be adapted for detection of different trigger oligonucleotides using the same set of DNA nanospheres. It permits detection of as low as 100 fM trigger oligonucleotide in under 10 min total assay time, with minimal reagent consumption and requirement for instrumentation. We expect that combining this simple, versatile assay with trigger generation from a genomic target DNA sequence of interest will be a powerful tool in the development of rapid and simple point-of-care molecular diagnostic applications. In the postgenomic era, improved methods for DNA detection hold great promise in enabling the rapid, sensitive, and accurate identification of clinical pathogens and biothreat agents, in the early detection of cancer and genetic diseases, and in genotyping of patients for personalized medicine.1-3 We have combined a new isothermal exponential nucleic acid amplification reaction (EXPAR) with visual, colorimetric detection mediated by DNAfunctionalized gold nanospheres to enable the detection of short DNA sequences in a simple and rapid manner with minimal requirement for instrumentation. Mirkin and co-workers have used gold nanospheres functionalized with a high density of oligonucleotides for sensitive nucleic * To whom correspondence should be addressed. E-mail: [email protected]. Tel.: (909) 607-9854. † Keck Graduate Institute of Applied Life Sciences. ‡ Harvey Mudd College. (1) Amos, J.; Patnaik, M. Hum. Mutat. 2002, 19, 324-33. (2) McGlennen, R. C. Clin. Chem. 2001, 47, 393-402. (3) van Belkum, A. Curr. Opin. Pharmacol. 2003, 3, 497-501.

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acid detection.4-7 Solution-based assays involve two sets of DNA nanospheres, each bearing a distinct probe sequence immobilized at the 5′- and 3′-terminus, respectively. Hybridization of these DNA nanospheres to a bridging DNA sequence causes formation of large three-dimensional aggregates and a shift of the gold nanosphere’s plasmon resonance band resulting in a red-to-blue color change of the solution. Through spotting the solution on C18-modified silica thin-layer chromatography (TLC) plates,5,7 the color change is sufficiently enhanced to permit direct visual readout. Alternatively, aggregation of gold nanoparticles can be detected based on colorimetric light scattering.8 Spot test detection of DNA is, however, not sensitive enough for diagnostic applications without amplification. Coupling of spot test detection with the polymerase chain reaction (PCR) has been demonstrated,9,10 but PCR requires extensive sample preparation, thermocycling equipment, and often lengthy thermocycling protocols. PCR amplicons are also longer than ideal for this type of assay, giving rise to a slower response time and less color change compared to short bridging trigger sequences.6 Mirkin and co-workers have developed alternative PCR-less detection schemes based on nanoparticle-catalyzed reduction of silver on surfaces, with subsequent readout based on absorbance,11 light scattering,12 changes in conductivity,13 or surface-enhanced Raman scattering.14 The sensitivity of these assays can be further enhanced through a multiparticle “bio-bar-code” approach.15 Detection involving silver (4) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-81. (5) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959-64. (6) Storhoff, J. J.; Lazarides, A. A.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L.; Schatz, G. C. J. Am. Chem. Soc. 2000, 122, 4640-50. (7) Jin, R. C.; Wu, G.; Li, Z.; Mirkin, C. A.; Schatz, G. J. Am. Chem. Soc. 2003, 125, 1643-54. (8) Storhoff, J. J.; Lucas, A. D.; Garimella, V.; Bao, Y. P.; Muller, U. R. Nat. Biotechnol. 2004, 22, 883-7. (9) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J.; Elghanian, R.; Taton, T. A. Nanoparticles having oligonucleotides attached thereto and uses therefor. Nanospere, Inc. U.S. Patent 6,730,269, 2004. (10) Viswanadham, G.; Elghanian, R.; Mirkin, C. A.; Letsinger, R. L. Abstr. Pap. Am. Chem. Soc. 1999, 218, U123. (11) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 1757-60. (12) Storhoff, J. J.; Marla, S. S.; Bao, P.; Hagenow, S.; Mehta, H.; Lucas, A.; Garimella, V.; Patno, T.; Buckingham, W.; Cork, W.; Muller, U. R. Biosens. Bioelectron. 2004, 19, 875-83. (13) Park, S. J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 1503-6. (14) Cao, Y. W. C.; Jin, R. C.; Mirkin, C. A. Science 2002, 297, 1536-40. (15) Nam, J. M.; Stoeva, S. I.; Mirkin, C. A. J. Am. Chem. Soc. 2004, 126, 59323. 10.1021/ac051364i CCC: $30.25

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reduction is highly sensitive but requires additional steps, which increases the total assay time and complexity. Our approach utilizes the simple visual colorimetric detection of DNA nanosphere aggregates through spotting of the solution onto a C18-TLC plate, which can be performed rapidly and requires minimal instrumentation. To address the sensitivity limitations, we take advantage of EXPAR, a new exponential nucleic acid amplification reaction, which involves a combination of polymerase strand extension and single strand nicking.16 Unlike PCR, EXPAR rapidly amplifies short oligonucleotides 106-109 fold through an isothermal reaction at moderate temperatures in a manner of minutes and is therefore ideally suited for interfacing with DNA-colloid hybridization. The EXPAR reaction amplifies a 10-20-bp trigger oligonucleotide X, which ultimately is to be generated from a genomic DNA target sequence of interest. The trigger oligonucleotide X primes an amplification template consisting of two times the complimentary sequence X′ separated by nine bases that will enable generation of a nicking enzyme recognition and cleavage site as depicted in Figure 1. The reaction occurs at 55 °C, a temperature that permits activity and stability of the two enzymes involved in the reaction, a thermophilic polymerase and the nicking enzyme N.BstNBI.17,18 This temperature is above the Tm for the short trigger oligonucleotide X, which only transiently primes the amplification template. However, upon extension by the polymerase, the elongated duplex becomes thermodynamically stable. Extension further creates the recognition site for the nicking enzyme, which nicks the top strand and thereby creates another identical trigger oligonucleotide that is thermodynamically unstable and melts off the amplification template. The polymerase elongates the recessed 3′-hydroxyl, and another trigger is created in the same manner. These trigger oligonucleotides then prime other amplification templates, creating true chain (exponential) reactions. We have developed a novel two-stage EXPAR amplification scheme (Figure 2), which involves two templates: the first template termed X′-X′ enables amplification of the trigger oligonucleotide X. The second template termed X′-Y′ enables the conversion of trigger X to a reporter oligonucleotide Y. This reporter oligonucleotide subsequently hybridizes to and aggregates a set of DNA-functionalized gold nanospheres, each bearing half of the complementary sequence immobilized at the 5′- and 3′-terminus, respectively. By combining this assay with spotting of the test solution onto a C18-modified silica plate, the (16) Van Ness, J.; Van Ness, L. K.; Galas, D. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4504-9. (17) The “official” designation of N.BstNBI as defined in REBASE, the restriction enzyme database has changed from N.BstNBI to Nt.BstNBI, where the additional “t” indicates cleavage of the top strand of the recognition sequence. This change was made to conform to the reconfigured nomenclature for restriction enzymes detailed in the subsequent reference. (18) Roberts, R. J.; Belfort, M.; Bestor, T.; Bhagwat, A. S.; Bickle, T. A.; Bitinaite, J.; Blumenthal, R. M.; Degtyarev, S. K.; Dryden, D. T. F.; Dybvig, K.; Firman, K.; Gromova, E. S.; Gumport, R. I.; Halford, S. E.; Hattman, S.; Heitman, J.; Hornby, D. P.; Janulaitis, A.; Jeltsch, A.; Josephsen, J.; Kiss, A.; Klaenhammer, T. R.; Kobayashi, I.; Kong, H. M.; Kruger, D. H.; Lacks, S.; Marinus, M. G.; Miyahara, M.; Morgan, R. D.; Murray, N. E.; Nagaraja, V.; Piekarowicz, A.; Pingoud, A.; Raleigh, E.; Rao, D. N.; Reich, N.; Repin, V. E.; Selker, E. U.; Shaw, P. C.; Stein, D. C.; Stoddard, B. L.; Szybalski, W.; Trautner, T. A.; Van Etten, J. L.; Vitor, J. M. B.; Wilson, G. G.; Xu, S. Y. Nucleic Acids Res. 2003, 31, 1805-12.

Figure 1. Overview of the exponential amplification reaction EXPAR. (A) a short trigger oligonucleotide transiently binds to the complementary recognition sequence at the 3′-end of the amplification template; (B) the trigger sequence is extended through the action of a DNA polymerase, forming the double-stranded nicking enzyme recognition site 5′-GAGTCNNNN-3′ on the top strand; (C) the top strand is cleaved through the nicking endonuclease N.BstNBI; (D) at the temperature of the reaction (55 °C), the newly formed trigger is released from the amplification template; additional trigger oligonucleotides are generated in the linear amplification reaction comprising duplex extension, nicking, and release; (E) the newly formed trigger oligonucleotides activate additional template sequences, giving rise to exponential amplification of the trigger X.

presence or absence of trigger oligonucleotide X can be detected visually. This two-stage EXPAR scheme allows for a flexible assay design, since the trigger sequence X is completely independent from the bridging reporter oligonucleotide sequence Y. It enables the same set of DNA nanospheres to be used for detection of any trigger X, eliminates the effort and cost involved in having to resynthesize DNA-functionalized gold nanospheres with specific probe sequences for each new assay, and enables optimization of nanosphere hybridization independent from the sequence to be detected. Independence of X from Y will further be important in future efforts toward multiplexing of the assay. We herein demonstrate the sensitive, rapid, and sequencespecific detection of short synthetic trigger oligonucleotides by the two-stage EXPAR reaction coupled with DNA nanosphere aggregation. Trigger generation from genomic DNA will be the subject of a separate study. An assay combining trigger generation with rapid, isothermal amplification and a simple visual readout has the potential to become a practical and important Analytical Chemistry, Vol. 77, No. 24, December 15, 2005

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Figure 2. Overview of the two-stage EXPAR reaction with detection through DNA-Au nanosphere hybridization: (A) exponential amplification of trigger X; (B) conversion of trigger X to reporter Y; (C) DNA-Au nanosphere aggregation mediated by the bridging reporter Y.

tool for point-of-care clinical diagnostics applications and biothreat detection. EXPERIMENTAL SECTION Nuclease-free water and 2 M KCl were purchased from Ambion (Austin, TX). Thiol-modified oligonucleotides in disulfide-protected form and amplification templates were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). N.BstNBI nicking enzyme, Bst polymerase, 10× Thermopol II buffer (Mg-free), and 100 mM MgSO4 were purchased from New England Biolabs (Beverly, MA). Other reagents were purchased through Fisher Scientific (Hampton, NH) or Sigma-Aldrich (St. Louis, MO). NAP-5 columns were purchased from Amersham Biosciences (Piscataway, NJ). General EXPAR Protocol. EXPAR amplification reactions were carried out in a reaction mixture containing 0.4 unit/µL N.BstNBI nicking enzyme, 0.08 unit/µL Bst polymerase, 250 µM each dNTP, 8 mM MgSO4, 1.2 mM EGTA, 47.5 mM KCl, 30 mM Tris-HCl, 15 mM (NH4)2SO4, 0.5 mM DTT, and 0.2% Triton X-100. Unless otherwise noted, the master mix further contained 100 nM X′-X′ template for experiments wherein the trigger X was solely amplified and 100 nM X′-X′ template plus 400 nM X′-Y′ template for experiments wherein the trigger X was amplified and then converted to reporter Y. The master mix was prepared in two parts. Part A contained 0.8 unit/µL N.BstNBI nicking enzyme, 2.4 mM EGTA, 75 mM KCl, 20 mM Tris-HCl (pH 8.4), 10 mM (NH4)2SO4, 1 mM DTT, and 0.2% Triton X-100. Part B contained 0.16 unit/µL Bst polymerase, 500 µM each dNTP, and 16 mM MgSO4 in 2× Thermopol II buffer (20 mM KCl, 20 mM (NH4)2SO4, 40 mM Tris-HCl (pH 8.8), 0.2% Triton-X-100), plus the templates in 2× of their final concentration. Equal volumes of these two parts were then combined at 4 °C, aliquoted into PCR tubes, and the desired amount of trigger oligonucleotide was added to each tube. Reactions were conducted in 10-µL or 30-µL 7986

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volumes. For standard experiments, the tubes were heated to 55 °C for 1-4 min in an Eppendorf thermomixer R. For analysis by LC-ESI-MS, reactions were quenched at 4 °C. Fluorescence-Based Real-Time Monitoring of Trigger X Amplification. For real-time monitoring of trigger X amplification in the first stage of the two-stage EXPAR reaction, 1X SYBR Green II was added to the master mix and individual reactions were performed using a 10-fold trigger dilution series from 10 pM to 10 fM plus a negative control containing no trigger. Reactions were carried out in an MJ Research DNA Engine Opticon I (Waltham, MA) real-time thermocycler set to a constant temperature of 55 °C, and the fluorescence intensity was monitored in 10-s intervals over a period of 5 min. Mass Spectrometric Quantification of the Production of Trigger X and Reporter Y. To detect and quantify the amounts of trigger X and reporter Y produced in the first and second stage of the two-stage EXPAR reaction, amplified reaction mixtures were analyzed by LC-ESI-MS, using a Micromass LCT time-of-flight instrument (Manchester, U.K.) connected to an Agilent 1100 Series HPLC system (Palo Alto, CA). Buffer A was composed of 5 mM dimethylbutylamine acetate (DMBAA) in HPLC-grade water. Buffer B was composed of 5 mM DMBAA in 50% (v/v) water/acetonitrile. Samples were injected on a Waters Xterra MS column (C18 packing, 3.5 µM particle size, 125-Å pore size, 2.1 mm × 20 mm), heated to 30 °C, and separated using a gradient of 10-15% buffer B over 18 s, 15-30% buffer B over 2 min 12 s, followed by 30-90% buffer B over 30 s. The first 60 s of this gradient served as a desalting step, and the eluent was diverted to waste. MS spectra were acquired in electrospray negative ion mode, ranging from 800 to 2000 amu, 1-s scan time. Spectra reported herein represent the integral of the main peak observed in the total ion current chromatograms. Using Sequence Editor to determine the predicted charge states from the known

sequences of trigger X and reporter Y, the program MassLynx was then used to quantify the mass chromatogram peaks representing the respective oligonucleotides. Conversion from peak area to trigger X or reporter Y concentration was performed using a standard curve generated from injections of known concentrations of either X or Y. With the current LC-MS protocols, the detection limits for short oligonucleotides is ∼100 nM. We quantified the amount of trigger X produced in the first stage of the reaction using EXPAR master mix containing 100 nM X′-X′ template and a 10-fold trigger dilution series from 10 pM to 10 fM plus a negative control containing no trigger, following amplification for various times. To optimize the amount of reporter Y produced by the two-template amplification scheme, we conducted EXPAR amplification reactions using 1 pM trigger X, and various ratios of X′-X′ and X′-Y′ template, with total template concentrations of 100, 500, and 1000 nM. These experiments were conducted in a slightly different master mix (no EGTA, 1× Thermopol I buffer (contains 8 mM MgSO4) and 0.5× nicking enzyme buffer (75 mM KCl, 5 mM Tris-HCl, 5 mM MgCl2, 0.5 mM DTT), both from New England Biolabs). We also quantified the amount of reporter Y produced using the optimized buffer composition plus 100 nM X′-X′ template, 400 nM X′-Y′ template, and 1 pM trigger, for various amplification times. Simulation of Trigger Y Production Kinetics. In conjunction with the mass spectrometric quantification, we have simulated the effect of varying concentrations of the first and second templates on reporter Y production using the biochemical kinetics simulator GEPASI.19 The binding of trigger X to the X′-X′ or the X′-Y′ template was assumed to be reversible, with a KD of 10 µM, representing the transient duplex formation at 55 °C. The extension and nicking reactions were assumed to be irreversible with rate constants of 3 and 0.3 s-1, respectively. These values are similar to previously reported kinetic constants for the EXPAR reaction, conducted under slightly different experimental conditions.16 We carried out simulations for total template concentrations of 100, 500, and 1000 nM, varying the molar ratio of the X′-X′ template from 0 to 1 in increments of 0.05, with a total simulation time of 10 min. Synthesis of DNA-Au Nanospheres. Monodisperse 13-nm gold nanospheres were prepared by citrate reduction of HAuCl4 as previously described.5,20 Au nanosphere solutions were filtered through a Millex-GS 0.22-µm syringe filter (Millipore Corp), characterized by UV-visible spectroscopy using a SPECTRAmax PLUS 384 cuvette/plate reader (Molecular Devices, Sunnyvale, CA) and dynamic light scattering (DLS) using a Protein Solutions DynaPro-MS/X system (12-µL quartz sample cell, Proterion Corp., Piscataway, NJ), and used within a few days. DNA functionalization of Au nanospheres was performed according to literature procedures,7,21 with a few modifications: the starting solution of bare Au colloids was degassed on ice for 30 min under aspirator vacuum and then placed under argon at room temperature. During the salt aging process, 2 M NaCl solution was added with a syringe pump at the rate of 10 µL/min (19) Mendes, P. Trends Biochem. Sci. 1997, 22, 361-3. (20) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735-43. (21) Reynolds, R. A.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 2000, 122, 3795-6.

under constant stirring, and incubation periods following each addition of NaCl were lengthened to 12 h. After the final incubated period, the DNA-Au nanosphere solution was diluted 2-fold with TE buffer and transferred to a stirred ultrafiltration cell (50 mL, Millipore Corp.) with a 5-kDa MWCO ultrafiltration membrane (regenerated cellulose, Millipore Corp.). The DNA nanospheres were purified via diafiltration with TE buffer for 6 h, concentrated to 5-8 nM, and stored in TE buffer plus 0.01% sodium azide at 4 °C. The concentration of DNA-Au nanoparticles was determined by UV-visible spectroscopy (molar extinction coefficient, 2.7 × 108 M-1 cm-1 at λmax ) 520 nm).7 Monodispersity and average hydrodynamic radius were determined via DLS from the autocorrelation function of the scattered light of 25-40 readings using 10-30-s reading windows. DNA Nanosphere Hybridization in Solution. To test DNA nanosphere hybridization and aggregation mediated by reporter Y as a function of ionic strength,7 75 µL of a solution containing 2.5 nM each of the 3′- and 5′-DNA probe-modified DNA nanospheres was combined with an equal volume of different NaCl solutions, resulting in final NaCl concentrations ranging from 0 to 300 mM. To assess DNA nanosphere hybridization in the buffer used for our assay, 75 µL of a solution containing 2.5 nM each of the 3′- and 5′-DNA probe-modified DNA nanospheres, 300 mM NaCl, and between 0 and 15 mM MgSO4 was combined with an equal volume of the EXPAR master mix without templates and trigger oligonucleotides. Except for the negative controls, 1 µM reporter Y was added to each mixture. Solutions were heated to 55 °C for 10 min, incubated at room temperature for 1 h, and the extent of DNA nanosphere aggregation was assessed via UVvisible spectroscopy and DLS as described above. Subsequent experiments for spot test detection of DNA used a solution containing 300 mM NaCl, 7.5 mM MgSO4, and 2.5 nM of each DNA nanosphere in TE buffer, thereafter referred to as DNA nanosphere detection reagent. This solution was stable at 4 °C for >1 month. Spot Test Detection of DNA Nanosphere Hybridization. To determine the minimum concentration of reporter oligonucleotide Y necessary for nanosphere hybridization, we combined 10 µL of EXPAR master mix containing no trigger or templates, but externally added reporter oligonucleotide Y (2-fold dilution series from 800 to 50 nM as well as a negative control without Y) with an equal volume of the DNA nanosphere detection reagent. After incubation at room temperature for 5 min, ∼3-µL aliquots of these solutions were spotted onto a C-18 modified silica thin-layer chromatography plate (150-µm layer, Alltech Associates, Deerfield, IL) in duplicate. We performed the same experiments including 400 nM X′-Y′ template in the EXPAR master mix, to test for interference by this template. To demonstrate colorimetric detection of trigger oligonucleotide X amplified via the two-stage EXPAR reaction coupled with DNA nanosphere aggregation, 10 µL of EXPAR master mix containing 100 nM X′-X′ template, 400 nM X′-Y′ template, and either 10 pM, 1 pM, 100 fM, or no trigger X, in addition to one negative control with no trigger X and no templates, and one positive control containing 1 µM externally added reporter Y but no trigger X and no templates, were heated to 55 °C for 1-4 min depending on the desired detection limit. Solutions were then removed from the heating block followed by immediate addition Analytical Chemistry, Vol. 77, No. 24, December 15, 2005

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Figure 3. (A) Real-time fluorometric monitoring of the trigger amplification reaction for different starting concentrations of trigger X, in the presence of SYBR Green II (data not normalized, differences in initial and final fluorescence intensities are due to well-to-well variations of the instrument). (B) Linear relationship between the inflection points of the fluorometric amplification curves and the log of the starting trigger concentrations. Data represent the average and standard deviation of six experiments conducted on three different days. (C) Concentration of amplified trigger X generated as a function of amplification time for different starting trigger concentrations, quantified by LC-ESI-MS.

of an equal volume of the DNA nanosphere detection reagent, incubation at room temperature for 2 min, and spotting as described above. For publication, the TLC plates of the spotting experiments were scanned using a Hewlett-Packard ScanJet ADF color scanner. The raw scanned images were found to be faded relative to the original. Using Adobe Photoshop 8.0, we therefore enhanced the image quality by applying the “Auto Color” and “Auto Contrast” functions with the default settings to the entire raw scanned images. These processes do not bias the color levels of one area of the image relative to another and, therefore, do not affect the relative color change observed between the spotted samples. Areas of the image containing the relevant information were then cropped and moved adjacent to one another. RESULTS AND DISCUSSION We report a novel approach for simple, rapid, and sensitive DNA detection, which involves the isothermal amplification of a synthetic trigger oligonucleotide X, the conversion of trigger X into a reporter oligonucleotide Y, and the visual colorimetric detection based on reporter Y-mediated DNA nanosphere aggregation. The following paragraphs discuss each step individually and then describe the combination of all steps into the overall assay. Amplification of Trigger X. The amplification of trigger X in the first step of the two-stage EXPAR reaction, using only the X′7988

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X′ template, can be followed in real time using a SYBR Green II-based assay that monitors the conversion of the X′-X′ template from the single-stranded into the partially double-stranded trigger producing form, giving rise to a sigmoidal curve (Figure 3A). Although generally used as a dye for detection of RNA or singlestranded DNA, SYBR Green II detects double-stranded DNA with a sensitivity comparable to SYBR Green I, and with higher sensitivity than single-stranded DNA, especially for short sequences.22 As opposed to SYBR Green I, SYBR Green II was found to not interfere with the EXPAR amplification reaction in a homogeneous format. The SYBR Green II assay demonstrates that amplification occurs rapidly and that the onset of efficient amplification is a linear function of the log of the starting trigger concentration in the range from 10 pM to 10 fM (Figure 3B). Onset of efficient amplification is here defined as the inflection point of the curves, i.e., the time at which ∼50% of the template is converted into the partially double-stranded, trigger producing form. Using the reported conditions and enzymes, background amplification in the absence of trigger is observed for reaction times of 5 min and longer. We are investigating the cause of this background amplification phenomenon. The SYBR Green-based assay only indirectly implies the amplification of trigger X. Through LC-ESI-MS, we have unam(22) Stothard, J. R.; Frame, I. A.; Miles, M. A. Anal. Biochem. 1997, 253, 2624.

Figure 4. LC-ESI-MS spectra of (A) trigger oligonucleotide X (expected m/z: 1047.30 (-3) and 1572.00 (-2)) produced in the first step and (B) reporter oligonucleotide Y (expected m/z: 1226.79 (-4) and 1636.39 (-3)) produced in the second step of the two-stage EXPAR reaction. Total amount of reporter Y produced by the two-stage EXPAR reaction starting from 1 pM trigger X in 10 min as a function of template molar ratio, using a 500 nM total template concentration (C) experimentally determined through LC-ESI-MS and (D) numerically simulated using the software package GEPASI.19

biguously identified the amplified trigger X based on its m/z ratio (Figure 4A) and have quantified the amount of trigger produced as a function of starting trigger concentration and amplification time (Figure 3C). The LC-MS chromatograms also demonstrate that the reaction produces trigger X in relatively pure form. For reactions containing 10 pM-100 fM starting trigger oligonucleotide, the time points at which significant trigger amplification (>200 nM) can be detected by MS roughly correlate with the time at which the maximum plateau is reached in the SYBR assay. Within the 3-min time frame of this experiment, reactions containing 10 fM starting trigger and the negative controls without trigger did not produce amplified trigger above the MS detection limit of 100 nM. Conversion of Trigger X to Reporter Y. The amplification of trigger X can be coupled to conversion to reporter Y by including the X′-Y′ template into the EXPAR reaction. Since the SYBR Green-based assay cannot deconvolute the two stages, we have used LC-ESI-MS combined with numerical simulations to characterize and optimize this reaction. Through LC-ESI-MS, we have confirmed the production of reporter Y through the twostage EXPAR reaction (Figure 4B). Again, the reaction produces reporter Y in relatively pure form according to the LC-MS chromatograms. We have quantified the amount of reporter Y produced as a function of the total template concentration and template ratio, both experimentally (Figure 4C) and through numerical simulations using the software package GEPASI (Figure 4D).19 The yield of reporter Y was found to be particularly sensitive to the template ratio. Efficient production of reporter Y is both predicted by simulations and experimentally observed using a molar ratio of the first template (X′-X′) between 0.2 and 0.4 for a total template concentration of 500 nM. At higher molar ratios of the first template, the trigger oligonucleotide X is sufficiently amplified but not effectively converted to reporter Y. At lower

molar ratios of the first template, most of the trigger X becomes sequestered by the second template, which effectively abolishes amplification of trigger X. Analogous experiments and simulations for 100 and 1000 nM total template concentrations reveal the same qualitative shapes of the curves (data not shown). Experimental and simulated results are in good agreement concerning the relative dependency of trigger production on the ratio of the two templates, but in all cases, the simulations overestimate the absolute amount of trigger Y produced and erroneously predict a significant increase in the yield of reporter Y upon increasing the total template concentration. Experiments show a much smaller and nonlinear dependency of reporter Y production on total template concentration. In subsequent experiments, we have used a total template concentration of 500 nM and a 0.2 molar ratio of the X′-X′ template. With these template concentrations and the optimized buffer, we have generated 1.3 µM reporter Y from 1 pM trigger X in 2 min as determined by LC-ESI MS, a larger than 106-fold amplification/conversion yield. DNA-Au Nanosphere Hybridization under EXPAR Conditions. Bare and DNA-functionalized 13-nm gold nanospheres were synthesized in high yield and large quantity, and were characterized by DLS and UV-visible spectroscopy (Figure 5A). These particles were found to be monodisperse with the expected plasmon resonance λmax. The increase in hydrodynamic diameter between bare and DNA-functionalized gold nanospheres with no increase in polydispersity and the stability of DNA-Au nanospheres in high ionic strength buffer are indicators for successful DNA modification. We have characterized the aggregation of DNA-functionalized nanospheres in solution mediated by external addition of bridging reporter oligonucleotide Y through the resulting increase in average diameter and polydispersity measured via DLS, and the shift of plasmon resonance maximum to longer wavelengths Analytical Chemistry, Vol. 77, No. 24, December 15, 2005

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Figure 6. Aggregation of DNA nanospheres, visualized by a color change from red to blue or purple, as a function of the concentration and length of reporter oligonucleotide Y, as well as competition caused by inclusion of the second template (X′-Y′) of the two-stage EXPAR reaction. Experiments entailed addition of 10 µL of DNA nanosphere detection reagent to 10 µL of EXPAR master mix containing trigger and template oligonucleotides, followed by room temperature incubation for 2 min and spotting on a C18 modified reversed-phase silica plate. Each solution was spotted in duplicate.

Figure 5. (A) Average hydrodynamic diameter and plasmon resonance maximum of bare and DNA-functionalized Au nanospheres characterized by DLS and UV-visible spectroscopy. (B) Increase in aggregate diameter and plasmon resonance maximum caused by aggregation of mixtures containing 3′- and 5′-probe-functionalized DNA nanospheres, mediated by the addition of 1 µM reporter oligonucleotide Y as a function of NaCl concentration. (C) Analogous experiments under EXPAR conditions: Aggregation of a solution containing equimolar amounts of DNA nanospheres functionalized with the 3′- and 5′-DNA probe sequences in 300 mM NaCl and between 0 and 15 mM MgSO4, combined with an equal volume of the EXPAR master mix containing reporter Y (1 µM final concentration) but no templates and trigger. For (B) and (C), solutions were heated to 55C for 10 min and subsequently analyzed. Each series includes negative controls with no trigger added.

measured via UV-visible spectroscopy. Experiments performed in buffer containing different concentrations of NaCl (Figure 5B) show that as expected the aggregation is strongly dependent on ionic strength. No aggregation is observed in 300 mM NaCl in the absence of reporter, which verifies the specificity of the process. We performed similar studies under the buffer conditions to be used for combining DNA nanosphere aggregations with the two-stage EXPAR amplification (Figure 5C), using an EXPAR master mix containing externally added reporter Y but no templates and trigger. As before, specific DNA nanosphere aggregation is observed from both the DLS and UV-visible data, albeit to a smaller extent, which can be attributed to the overall change in buffer composition. The hybridization efficiency is improved by the addition of magnesium. Based on these results, subsequent experiments were conducted using a stock solution referred to as DNA nanosphere detection reagent, containing both DNA nanospheres, NaCl, and MgSO4 at concentrations conducive to stable storage for 1 month or longer at 4 °C. We visually detected the DNA nanosphere aggregation mediated by external addition of reporter Y through the spot test (Figure 6).4,5 We found that after adding the DNA nanosphere detection reagent to the EXPAR master mix, a definitive readout marked by a color change from red to dark purple or blue can be obtained without heating the mixture and using a room temperature incubation period of only 2 min. We have scaled down the volumes per reaction to 10 µL each of the EXPAR master mix 7990 Analytical Chemistry, Vol. 77, No. 24, December 15, 2005

and DNA nanosphere detection reagent, and further reduction is possible. This translates into significantly reduced reagent consumption per sample. Through this spot test we have investigated how the length of reporter oligonucleotide Y affects the efficiency of gold nanosphere aggregation (see Table 1 for sequences used). In the absence of the second template, the 24-mer reporter sequence hybridizes to and aggregates the DNA nanospheres more effectively than the shorter 20- or 16-mer reporter sequences. In the presence of 400 nM concentrations of the second (X′-Y′) template, the DNA nanospheres have to compete for binding of the reporter oligonucleotide Y, which results in an increase of the minimal reporter concentration required for effective DNA nanosphere aggregation to ∼800 nM, irrespective of reporter length. Through the twostage EXPAR reaction, we are however able to generate >1 µM reporter Y in a few minutes. Therefore, the sensitivity of the assay is mainly provided by the amplification of trigger X but may be further improved by optimizing the sensitivity of the reporter Y-mediated DNA nanosphere aggregation. We selected a 16-mer reporter oligonucleotide for the overall assay since it is efficiently generated by the two-stage EXPAR reaction, but longer reporter sequences can be generated by the EXPAR reaction as well. EXPAR Amplification Combined with Detection through DNA Nanosphere Aggregation. The final assay combines the two-stage EXPAR reaction with DNA nanosphere-based visual detection in the following format: the trigger oligonucleotide X is amplified and converted to reporter oligonucleotide Y in the EXPAR master mix at 55 °C for 1.5-3.5 min, dependent on the desired detection limit (Figure 7A). The amplified reaction mixture is then removed from the heating block and combined with an equal volume of the DNA nanosphere detection reagent. After another 2 min of incubation at room temperature, the solution is spotted onto the TLC plate, resulting in a purple/blue spot as a positive readout or a red spot as a negative readout. Using this format, the overall assay can be conducted in 10 min or less. The assay contains a total of three controls: the controls labeled “positive” and “negative” contain no template and trigger oligonucleotides and either 1 µM externally added reporter or no reporter oligonucleotide, respectively. These controls verify that DNA nanosphere aggregation occurs in a specific manner, caused by the presence of the bridging reporter oligonucleotide

Table 1. Oligonucleotide Sequences Used name

sequence

5′-probe 3′-probe 24-mer reporter 20-mer reporter 16-mer reporter (Y) trigger X (A) X′-X′ template (A) X′-Y′ template (A) trigger X (B) X′-X′ template (B) X′-Y′ template (B)

5′-HS-TTT TTT TTT CGG TCT GGC GCT-PO3-3′ 5′-TGA TGG TAC GGG TTT TTT TTT-SH-3′ 5′-CCC GTA CCA TCA AGC GCC AGA CCG-3′ 5′-C GTA CCA TCA AGC GCC AGA C-3′ 5′-TA CCA TCA AGC GCC AG-3′ 5′-CAG TCG TAG G-3′ 5′-CCT ACG ACT GAA CAG ACT CTC CTA CGA CTG-NH2-3′ 5′-CTG GCG CTT GAT GGT ATC CAG ACT CTC CTA CGA CTG-NH2-3′ 5′-GAG ACT GCA G-3′ 5′-CTG CAG TCT CAA CAG ACT CTC TGC AGT CTC-NH2-3′ 5′-CTG GCG CTT GAT GGT ATC CAG ACT CTC TGC AGT CTC-NH2-3′

trigger B-specific set of templates, a positive result is obtained only in the presence of trigger B. This verifies that the assay can readily be redesigned to detect a different trigger sequence simply by choosing a different set of appropriate template oligonucleotides. While these two sets of templates amplify different triggers, both triggers are converted to the same bridging reporter sequence Y. This enables us to use the same set of DNA nanospheres irrespective of the trigger sequence to be detected, without the need for lengthy resynthesis of DNA nanospheres with appropriate probe sequences or reoptimization of assay conditions. It further allows us to optimize the reporter sequence and probe sequences on the DNA nanospheres independent from the trigger sequence to be detected.

Figure 7. Visual detection of trigger oligonucleotide (positive, blue/ purple; negative, red) by the two-stage EXPAR reaction combined with DNA nanosphere-based colorimetric detection through the spot test. (A) Sensitivity: semiquantitative detection of trigger oligonucleotide concentrations between 10 pM and 100 fM through variation of the amplification time. (B) Specificity: discrimination between two different trigger sequences through appropriate template sequences.

as opposed to nonspecific aggregation caused by instability of the DNA nanospheres under the assay conditions. The assay contains a second negative control labeled “no trigger”, which contains the template oligonucleotides but no trigger or externally added reporter oligonucleotides. This control verifies the specific generation of the reporter Y by the two-stage EXPAR reaction caused by the presence of trigger X, the analyte of interest. To verify sequence-specific DNA detection, we have performed the assay with two different sets of templates: one set designed for the standard trigger used thus far, here called trigger A, the other set designed for a different trigger oligonucleotide, here called trigger B (see Table 1 for sequences). As shown in Figure 7B, using the trigger A-specific set of templates, a positive result is obtained only in the presence of trigger A. Likewise, using the

CONCLUSIONS We have developed a rapid, semiquantitative, sequence-specific method for detecting short oligonucleotides that combines a novel isothermal DNA amplification method with detection through aggregation of DNA-functionalized gold nanospheres. The overall assay can be conducted in 10 min or less, with minimal reagent consumption and simple visual colorimetric detection. The only instrumentation required is a heating block set to 55 °C. The sensitivity of our assay compares favorably with previous reports of DNA detection based on Au nanosphere aggregation in solution. The lower limit for the visual colorimetric detection of a bridging oligonucleotide by the spot test has been reported to be ∼10 nM (10 fmol; i.e., 6 × 109 copies in 1 µL).4,23 As an alternative to absorbance-based detection through spotting on TLC plates, researchers at Nanosphere Inc. have developed a system based on colorimetric scattering of aggregated DNA nanospheres spotted onto a glass surface.8 This approach increases the sensitivity to 33 pM (166 amol; i.e., 108 copies in 5 µL) but requires a 2-h hybridization step and more complex optical instrumentation. In our studies, the amount of reporter Y required for DNA nanosphere aggregation detectable by the spot test is much higher (Figure 6), due to the shorter overlap length between probe and reporter sequences, the composition of the EXPAR buffer, and the shorter hybridization time. It is, however, important to note (23) Different articles adopt different conventions for reporting sensitivity. The herein listed sensitivities are based on the concentration and volume of the DNA-containing solution prior to addition of DNA nanospheres and hybridization buffer. Likewise, sensitivity in this report refers to the concentration of analyte DNA in the EXPAR master mix, prior to addition of the DNA nanosphere detection reagent. We use concentration as the primary indicator for sensitivity, since it is independent of total assay volume as opposed to molar amount or DNA copy number.

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that detection of reporter Y is an intermediate step in the assay and does not reflect the overall sensitivity for detection of trigger oligonucleotide X, the analyte of interest. Through coupling of DNA nanosphere aggregation with the two-stage EXPAR reaction, we can detect 100 fM trigger X (1 amol; i.e., 6 × 105 copies in 10 µL) through a short and simple overall assay. This represents a significant increase in sensitivity compared to literature reports. The sensitivity of DNA detection mediated by DNA-Au nanospheres has been increased through hybridizing the bridging target and DNA nanospheres to a probe-functionalized surface, followed by signal amplification through Au nanosphere-catalyzed silver reduction and scanometric detection.11,12 The original report11 states a sensitivity of 50 fM, but the assay requires overnight hybridization, washing, 15-min developing through silver reduction, washing, and scanometric detection. An improved system has been developed, which achieves comparable sensitivity (100 fM in 50 µL, i.e., 3 × 106 copies) using a 1-h hybridization and a 5-min silver reduction step.12 With our assay, we are able to detect the trigger oligonucleotide X with comparable sensitivity (100 fM in 10 µL, i.e., 6 × 105 copies) through a simpler and much faster overall process. However, researchers at Nanosphere Inc. have demonstrated detection of sheared genomic DNA with only slightly lower sensitivity (200 fM in 50 µL, i.e., 6 × 106 copies), an important milestone that still needs to be demonstrated using our system. In addition, through the bio-bar-code approach,15 the sensitivity of DNA detection through Au nanosphere-catalyzed silver reduction can be further improved to reach PCR-like levels (10 copies in 30 µL), however, at the expense of requiring several additional assay steps, which prolongs and complicates the overall procedure. The simplicity and reasonably high sensitivity of the herein reported assay may facilitate implementation in a point of care setting. To achieve this goal, we will expand upon our proof-ofprinciple results, specifically addressing three key points. First, the amplification and detection reactions have to be coupled with trigger generation from genomic target DNA. Trigger generation from genomic DNA or RNA has been demonstrated via several approaches developed by the laboratory of Dr. Galas and Ionian Technologies.24,25 One approach is based on the existence of adjacent nicking enzyme recognition sites in genomic DNA, which occurs at a frequency of ∼1/106 base pairs, thus enabling the generation of a distinct set of trigger oligonucleotides specific for each host organism. Trigger generation from genomic DNA by this approach has been observed experimentally using mass spectrometry. In silico analysis shows very little overlap between different sets of trigger oligonucleotides predicted to be generated from the genomes of even closely related organisms, such as the human herpesviridae HHV1-HHV8. The entire motif required for trigger generation includes the trigger sequence plus flanking (24) Van Ness, J.; Galas, D. J.; Van Ness, L. K. Exponential nucleic acid amplification using nicking endonucleases. Keck Graduate Institute. U.S. Patent application 20030082590, 2005. (25) VanNess, J.; Galas, D. J. Keck Graduate Institute, 05/15/2005, personal communication.

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nicking enzyme recognition sites, which increases the total length and results in increased specificity. This approach, however, only enables trigger generation at fixed sites and may not permit detection of arbitrary sequences or specific single-nucleotide polymorphisms. This limitation is addressed through an alternative approach that uses specific hybridization probes, which generate triggers in the presence of a single-stranded genomic DNA or RNA sequence of interest. This approach enables trigger generation from any arbitrary sequence and has been successfully applied to SNP discrimination. As a second key point, the sensitivity of the overall reaction needs to be within clinically relevant ranges. Currently, we can detect 100 fM trigger oligonucleotide in a 10-µL reaction volume, equal to ∼6 × 105 copies of trigger. In one application, the DNAbased detection of herpes simplex virus (HSV) in swab samples of herpetic lesions, PCR-based assays have shown the viral target DNA to be present at an average of 104 copies/reaction.26 HSV diagnosis in cerebrospinal fluid requires detection of 102 copies of target DNA or less per reaction,27 but this procedure would not be performed in a point of care setting. The sensitivity of our assay can be improved by optimizing the DNA nanosphere detection reagent, by suppressing or eliminating the background amplification within the EXPAR reaction, and by effectively taking advantage of the trigger generation reaction from genomic DNA, which involves linear amplification that can contribute 1-2 orders of magnitude to the overall sensitivity. As a final third point, the assay should be implemented in a format that avoids carryover contamination from one sample to the next, for example, by conducting the overall reaction in a disposable closed system microfluidic device. We anticipate that such a device capable of trigger generation from genomic target DNA, trigger amplification and conversion using the two-stage EXPAR reaction, and visual colorimetric detection based on DNA-functionalized gold nanospheres will be a valuable tool for clinical diagnostics applications and biothreat detection. ACKNOWLEDGMENT We thank Krisanu Bandyopadhyay for his role in the synthesis of DNA-functionalized gold nanospheres and their characterization by DLS. We thank Jeffrey Van Ness for helpful discussions and Annie Tan for her assistance in DNA nanosphere characterization. Funding for this project was provided by the Keck Graduate Institute and by the National Science Foundation through NER Grant BES-0304675 and REU Grant EEC-0243910.

Received for review August 1, 2005. Accepted October 4, 2005. AC051364I (26) Filen, F.; Strand, A.; Allard, A.; Blomberg, J.; Herrmann, B. Sex Transm. Dis. 2004, 31, 331-6. (27) Kimura, H.; Ito, Y.; Futamura, M.; Ando, Y.; Yabuta, Y.; Hoshino, Y.; Nishiyama, Y.; Morishima, T. J. Med. Virol. 2002, 67, 349-53.