Ultrafast Kinetic DNA Hybridization Assay Based on the Visualization

Mar 29, 2012 - identifiable, turbidity-definitive, and kinetic threshold state could be reached at a time as short as 1 min. The assay scheme allows f...
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Ultrafast Kinetic DNA Hybridization Assay Based on the Visualization of Threshold Turbidity Xin Shu, Jingzhi Lu, Haipeng Lv, Xu Zhang, Yishu Yan, Jingjing Sun, and Jin Zhu* Department of Polymer Science and Engineering, School of Chemistry and Chemical Engineering, State Key Laboratory of Coordination Chemistry, Nanjing National Laboratory of Microstructures, Nanjing University, Nanjing 210093, China S Supporting Information *

ABSTRACT: We report herein the development of an ultrafast kinetic DNA hybridization assay system based on the visualization of threshold turbidity associated with the assembly of polystyrene nanospheres. Initial testing of our diagnostic protocol on a sequence associated with the anthrax lethal factor indicates that a visually identifiable, turbidity-definitive, and kinetic threshold state could be reached at a time as short as 1 min. The assay scheme allows for both target concentration quantification and differentiation of single base mismatches through registry of the threshold turbidity onset time. The positively charged environment on nanospheres not only contributes to expedited signal generation but also imparts cooperative DNA binding properties. The kinetic visual protocol complements conventionally used thermodynamic strategies and provides an entry point for the circumvention of assay issues associated with ill-defined thermodynamic end points.

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NA hybridization,1−4 or sequence-specific DNA−DNA recognition, is an important assay tool for the examination of genetic information at the molecular level. The efficacy for the utility of such an indispensable molecular diagnostic technique is ultimately dictated by the capacity of the signal generation event. In this regard, assay systems based on conventional radioactive labels, fluorescent reporter probes, etc. have contributed enormously to the establishment of fundamental understanding of DNA binding kinetics and thermodynamics.5,6 The creative use of gold nanoparticle (AuNP)-derived surface plasmon signature from the Mirkin group has ushered in a paradigm shift for DNA hybridization assay.7−9 Indeed, the visual detection format has rendered previously infeasible point-of-care medical diagnostics a potentially achievable reality and therefore significantly impacted the way molecular diagnostics is practiced.10,11 In such a system, the visualization of red-to-blue colorimetric transition, characteristic of a change of certain-sized AuNPs from individually dispersed to aggregated state, indicates the occurrence of DNA hybridization. In spite of tremendous utility, the signal readout kinetics from such a nanoscale material structural transformation has yet to be further improved in order to bring the true benefit of fast and reliable testing to the biomedical community. Indeed, our own work in molecular diagnostics12−15 also indicates that the generation of colorimetric transition in AuNP-based systems typically requires an extended period of time. We reasoned that the slow signal generation associated with the AuNP system was an inherent character derived from both the heavy packing of negatively charged DNA and time-consuming achievement of threshold size for the requisite plasmon dampening and redshift. Therefore a distinct diagnostic architecture with fast © 2012 American Chemical Society

hybridization kinetics and better signal generation mode should be resorted to for the development of an expeditious assay platform. In the past few decades, immunoassays (including the detection of virus/bacteria16−19) based on particle agglutination processes have been developed. Various nanospheres have been synthesized to immobilize antigen or antibody by adsorption20,21 or covalent conjugation.22−27 The assays are achieved by either direct visual observation of particle aggregation28 or through the assistance of instrumentation (e.g., based on turbidimetry,29 nephelometry,30 angular anisotropy,31 photon correlation spectroscopy,32 particle number counting33). Although direct visual observation method is the most costeffective, the only quantification method developed is through serial dilutions of test fluids and identification of the disappearance of particle aggregation. The inconvenience associated with such a serial dilution strategy renders the visual assay format quite labor intensive. We reasoned that the structural features of DNA should ensure fundamentally distinct assay kinetics compared with proteins and other nonDNA targets, and thus an effective kinetic assay based on turbidity could be used for the circumvention of issues in thermodynamic end point analysis. Herein, we wish to report on the elaboration of an ultrafast kinetic DNA hybridization assay scheme through the visualization of threshold turbidity from the collective assembly and organization of nanospheres. In line with the Secchi depth method, the threshold turbidity is Received: October 7, 2011 Accepted: March 29, 2012 Published: March 29, 2012 3500

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turbidity assay enables the differentiation of perfect complementary DNA strand from DNA strands containing single base mismatches.

defined as the turbidity at which the image of a light source behind the sample could not be observed and the onset time of threshold turbidity is used to characterize the hybridization characteristics. Previous studies have indicated that various DNA bindingcapable reagents (e.g., heterogeneous nuclear ribonucleoprotein,34 cationic surfactants,35 comb-type cationic copolymer36) could contribute to the acceleration of DNA hybridization kinetics. The acceleration is furnished through the enhancement of DNA collision probability by virtue of either the transient interaction of DNA binding-capable reagents with DNA and/or reduction of electrostatic repulsion of negatively charged DNA. Therefore, we have chosen positively charged polystyrene nanospheres (PSNSs) as a demonstrating platform based on the following considerations: (1) Surface-functionalized PSNSs with controllable sizes could be facilely synthesized by emulsion polymerization of styrene in the presence of a minor amount of charge-imparting monomer (e.g., methacrylic acid,37,38 4-vinylbenzyl amine hydrochloride (4-VBAH),39−41 tbutoxycarbonyl-protected 4-aminostyrene42); (2) The amino functional groups on the PSNS surface would be useful for both the covalent conjugation of DNA43−46 and the generation of positively charged environment, thus facilitating electrostatic interaction between aminated PSNS (APSNS) surface and negatively charged DNA and increasing the likelihood of hybridization between complementary DNA strands; (3) The positive surface charge is also expected to reduce the electrostatic repulsion between negatively charged DNA strands and therefore enhance the collision probability of complementary strands. The expeditious hybridization kinetics of the APSNS system ensures the ability to achieve a fast DNA assay within the time frame of minutes. Besides the shortened assay time, an added advantage of our system is that the visual discernment of turbidity is inherently more straightforward than the visual resolution of subtle colorimetric transitions. In fact, because of the breadth of the surface plasmon bands, the colors of largersized AuNPs are essentially indistinguishable before and after the target-induced aggregation process.47 A spectrometer therefore has to be resorted to for readout under this circumstance, which renders it nonviable to use the assay protocol in resource-poor settings. We envisioned that the onset of kinetic threshold turbidity could be exploited for signifying DNA hybridization event. In contrast to many of the molecular diagnostic methods that rely on the thermodynamic end points for assay readout, our system is featured with a kinetically definable midpoint visual signature. Our assay protocol can be likened to quantitative real-time polymerase chain reaction (qPCR)48,49 in the sense that the threshold onset time, instead of the output signal intensity, is used as the value for characterizing target DNA. Therefore, besides the provision of an ultrafast readout scheme, the kinetic registration of DNA binding event with the visualization of threshold turbidity is especially useful for addressing the issues of illdefined or plateaued reaction course end points. Indeed, many of the diagnostic formats, like standard PCR, can not provide a meaningful interpretation of the target attributes (e.g., concentration) beyond the two-state, presence or absence judgment because of the reliance on end point analysis. The APSNS system is effectively applied in DNA diagnostics and provides a relatively wide dynamic quantification range of DNA concentrations compared with the thermodynamic end point analysis. Importantly, the onset time for threshold

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EXPERIMENTAL SECTION Materials. Reagents for organic synthesis were from commercial sources and used without further purification unless otherwise noted. All the DNA molecules (Supporting Information Table S1) used herein were custom-synthesized by Sangon Biotech (Shanghai) Co. Ltd. S1 nuclease was from TaKaRa Biotechnology (Dalian) Co. Ltd. Nanopure water (18.2 MΩ·cm), purified by Sartorius Arium 611 system, was used throughout the experiment. All the solvents were of analytical grade. Preparation and Characterization of APSNSs. APSNSs of two different diameters (∼195 and ∼353 nm) were prepared by copolymerization of styrene and 4-VBAH according to the previous reports.39,40 The concentration of APSNSs was determined by the measurement of weight for a dry APSNS sample in a certain volume of APSNS solution. N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was used to determine the concentration of surface amino groups of APSNSs (Supporting Information Scheme S1).41 SPDP solution in anhydrous 1,4-dioxane was added (with a total SPDP amount of A0) into the APSNS suspension (containing 0.15 v% Tween 20) in sodium bicarbonate buffer (SB buffer, 0.1 M, pH 8.2). The reaction of amino groups with SPDP was allowed to proceed at room temperature for 2 h under vortex conditions. The SPDP-modified APSNSs and unreacted SPDP were collected separately by centrifugation. To the two samples containing SPDP were added large amount of dithiothreitol (DTT) in order to reduce the disulfide bond to yield pyridine2-thione. If the amounts of pyridine-2-thione derived from SPDP-modified APSNSs and unreacted SPDP were measured (based on UV−vis absorption at 340 nm) as A1 and A2, respectively, the total amount of amino groups in the initial APSNS suspension could be calculated by the following equation: A( −NH 2) =

A1 × A0 A 2 + A1

Preparation of DNA-Conjugated APSNSs (DNAAPSNSs) and Determination of DNA Surface Coverage on DNA-APSNSs. 45,46 DNA1 or DNA2 (Supporting Information Table S1) (0.5 OD) was first mixed with an aqueous solution of sodium tetraborate (2 μL, 50 mM) and a N,N′-dimethyformamide (DMF) solution of 1,4-phenylene diisothiocyanate (DITC) (10 μL, 9 mg/mL). The reaction was allowed to proceed at room temperature for 12 h, followed by the precipitation of unreacted DITC with 88 μL of water. After centrifugation (15000 rpm, 5 min), the supernatant (DITC-modified DNA, 100 μL in total volume) was used for the modification of APSNSs. An aqueous-dispersed ∼195 nm APSNSs (200 μL) was mixed with an aqueous solution of sodium tetraborate (6 μL, 50 mM), 44 μL of water, and 50 μL of DITC-modified DNA. After being allowed to react at room temperature for 12 h, DNA-APSNSs were centrifuged, washed with water, and finally redispersed in 500 μL of 0.1 M NaCl, 10 mM phosphate buffered saline containing 0.15% Tween 20 (pH 7.0, designated PBSTW buffer). The final concentrations of as-prepared DNA1-APSNSs and DNA2-APSNSs are both 7.52 nM. 3501

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Note: DNA-APSNSs are unstable in PBS buffer solution, and therefore Tween 20 is added to stabilize the suspension. A fluorescent DNA strand is used to determine the DNA surface coverage on DNA-APSNSs. In particular, an aminated 6-carboxyfluorescein (FAM)-modified DNA strand was used in the surface modification of APSNSs. Direct measurement of the fluorescence of surface-confined DNA or measurement of the released fluorescent species after S1 nuclease cleavage allowed the determination of DNA surface coverage. General Procedure for the Measurement of Hybridization Properties of DNA-APSNSs. (1). Ultrafast Kinetic DNA Hybridization Assay Based on the Visualization of Threshold Turbidity. In a typical experiment, we kept the assay sample (inside a 1.5 mL Eppendorf tube) in front of a white light emitting diode lamp and observed the time-dependent variation of lamp image. Because of the hybridization-driven formation of APSNS assembly and the light scattering associated with the assembled structure, the assay sample would become turbid and the lamp image would become blurred. The threshold turbidity is defined as the turbidity at which the image of the lamp could not be observed. The direct visual identification is convenient and could provide important information for DNA through the registry of threshold turbidity onset time. The kinetic DNA hybridization assay was performed by the visual observation of time-dependent development of the turbidity in a DNA-APSNS suspension. Typically, 100 μL PBSTW solutions of DNA1-APSNSs (0.376 nM) and DNA2APSNSs (0.376 nM) were incubated in the presence of 1 μM complementary DNA3 (Supporting Information Table S1). The change of turbidity was continuously monitored. The onset time for threshold turbidity was identified to be 1 min in the presence of DNA3. (2). Melting Properties of DNA-APSNSs Hybridized with Target DNA. The melting properties of DNA-APSNS assembly structure formed through the hybridization with DNA3 were carried out by monitoring temperature-dependent change in the extinction value at 450 nm. Typically, aqueous-dispersed PBSTW solutions of DNA1-APSNSs (10 μL, 0.94 nM) and DNA2-APSNSs (10 μL, 0.94 nM) were mixed with 5 μL of 5 μM DNA3 in PBSTW buffer. The hybridization reaction was allowed to proceed at room temperature for 12 h. The sample was then diluted with 475 μL of PBSTW buffer for the measurement of temperature-dependent UV−vis extinction spectroscopy. (3). Characterization of DNA Hybridization-Driven DNAAPSNS Assembly Process by UV−vis Extinction Spectroscopy. Typically, aqueous-dispersed PBSTW solutions of DNA1APSNSs (10 μL, 0.94 nM) and DNA2-APSNSs (10 μL, 0.94 nM) were mixed with 5 μL of 5 μM, 0.5 μM, 50 nM, and 25 nM DNA3 in PBSTW buffer. Each sample was diluted by 475 μL of PBSTW buffer just before the measurement. The approximate threshold extinction value required for the visual identification of threshold turbidity could be facilely obtained. The threshold turbidity could be defined with the assistance of UV−vis extinction spectroscopy. The identification of threshold turbidity is achieved through the measurement of time-dependent extinction values of the assay sample at 450 nm, typically in a 1 cm cuvette. Based on the calibration with direct visual identification method, the threshold turbidity is defined as the turbidity at which the change in the extinction values against the assay starting point reaches 77% the total change (plateau value − starting value). This definition is valid

based on consistent observation of the threshold turbidity onset time at this 77% point in both direct visualization and macroscopic image formats.

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RESULTS AND DISCUSSION Our assay scheme relies on the utility of two batches of DNAAPSNSs, each containing a sequence capable of recognizing and aligning contiguously on target DNA (Figure 1). DNA

Figure 1. Schematic representation of kinetic DNA hybridization assay based on the visualization of threshold turbidity.

hybridization in the presence of target strand triggers a transparent-to-turbid transition by virtue of the DNA-APSNS structural transformation from individually dispersed to aggregated state. The ultrafast visual identification capability is ensured by the judicious selection of proper-sized APSNSs16,50−52 that possess close to transparent-to-turbid transition light scattering properties and therefore the ability to facilely cross the requisite size threshold (400−800 nm) to the turbid regime upon DNA binding. Sharp extinction changes (Mie theory for scattering) during the aggregation process would be observed if the term πd/λ is in the range of 1−2, where d and λ stand for the diameter of APSNSs and the wavelength of the incident light, respectively. For visualization at the visible wavelength (400 nm < λ < 800 nm), the proper diameter of APSNSs should fall in the range of 130−500 nm. Monodispersed APSNSs were synthesized with a radical emulsion polymerization system containing monomers of 4VBAH and styrene and an initiator of 2,2′-azobis(2amidinopropane) dihydrochloride (V50).39−41 The conjugation of DNA to APSNS surface was achieved through the reaction of aminated DNA and APSNSs with a bifunctional molecule, DITC. The as-prepared DNA-APSNSs, with an average diameter of ∼195 nm or ∼353 nm as characterized by scanning electron microscopy (SEM) (Supporting Information Figure S1), appear transparently brown (Figure 2A) if viewed in the transmittance mode, due to the light scattering properties. By quantifying the concentrations of APSNSs (18.8 nM) and surface amino groups (3.6 mM, Supporting Information Figures S2 and S3, Table S2),41 the average number of accessible amino groups on an ∼195 nm APSNS was determined to be 1.9 × 105, corresponding to ∼1.6 amino groups per square nanometer. In contrast to this, the average number of DNA molecules on a DNA-APSNS is merely 180 3502

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ensemble construct (Figure 3 and Supporting Information Figure S9).

Figure 2. Macroscopic transmittance images of DNA1-APSNSs and DNA2-APSNSs after incubation in the presence (left) and absence (right) of 1 μM DNA3 for 0 s (A), 30 s (B), 1 min (C), 2 min (D), 10 min (E), and 2 h (F). The onset time for threshold turbidity was identified to be 1 min. Figure 3. SEM images of a mixture of DNA1-APSNSs (10 μL, 0.94 nM) and DNA2-APSNSs (10 μL, 0.94 nM) after incubation with 5 μL of 5 μM DNA3 in PBSTW buffer for 0 s (A), 30 s (B), 1 min (C), 2 min (D), 10 min (E), and 2 h (F). All the samples were diluted to 1/5 of the original concentration when drop-casting onto the silicon wafers.

(Supporting Information Figures S4−S7, Tables S3 and S4), translating to the functionalization of 1 DNA strand per 1055 amino groups. Such a high density of amino groups, coupled with sparsely distributed DNA, should enable the creation of a highly positively charged environment for hybridization reaction when placed in an amino protonation-capable buffer conditions, as could be seen from the ζ potential (+50.3 mV for DNA1-APSNSs, Supporting Information Table S5). Considering the drastic DNA hybridization rate enhancement by cationic detergents,35 we reasoned that the high loading of positive charge could be beneficial to the DNA binding event because of the electrostatic interaction-driven increase of the collision probability of complementary strands. The initial evaluation of the feasibility of our system for the ultrafast kinetic DNA hybridization assay indeed confirms that the visualization of threshold turbidity could be rapidly achieved. To generate presentable data on the time-dependent variation of turbidity, the image of the assay sample could be permanently recorded through photography, with magnesium lamp placed behind a PCR tube as the light source. Photographs of samples are collected continuously at different times throughout the whole process. In this format, the threshold turbidity is defined as the turbidity at which the clear boundary generated by the refracted light on the side of PCR tube disappears. The corresponding onset time is measured and determined at this point. Thus, the mixing of DNA1-APSNSs and DNA2-APSNSs with the complementary target DNA3, a sequence associated with the anthrax lethal factor (1 μM),12−15 leads to the visually identifiable, turbidity-definitive, kinetic threshold state at a time as short as 1 min (Figure 2). At an elevated salt concentration (0.3 M NaCl), the time could be even shorter (45 s, Supporting Information Figure S8), while obvious color change from red to purple could only be observed after 2 h for DNA-conjugated AuNPs (DNA-AuNPs). The turbidity state after the threshold point does not provide any significant and valuable distinguishing feature except that at the far end of the structural organization process, extensive aggregation results in the precipitation of DNA-APSNSs out of the solution (Figure 2F). SEM allows for the observation of the evolution process of DNA-APSNS assembly into larger-sized

Although the hybridization kinetic rate constant at this DNA surface coverage was about 1.3 × 104 M−1·s−1 (Supporting Information Table S6 and Figure S10), the signal generation process is still fast enough due to the desired size and concentration selected for APSNSs. Three lines of evidence support the hypothesis that DNA hybridization is the driving force behind the assembly of DNA-APSNSs: (1) No visually identifiable aggregation process is observed for a control experiment carried out with a noncomplementary DNA4 (Supporting Information Table S1 and Figure S11); (2) A heating and cooling cycle across the melting temperature of DNA duplex performed on the target-linked DNA-APSNS aggregates leads to the turbid-to-transparent and transparentto-turbid transitions; (3) A sharp melting transition (melting temperature = 36 °C, Supporting Information Figure S12) could be observed on the UV−vis extinction spectroscopy, with an extremely narrow temperature range for melting (full-widthat-half-maximum, or fwhm =1.5 °C). The comparable sharpness to that derived from DNA-AuNPs53 not only validates the notion that DNA hybridization is essential for the assembly but also expands on the architecture format for the creation of sharp melting transition system. Most probably, the cooperative dehybridization properties are imparted by the collective interactions based upon weak attractive electrostatic forces and strong DNA hybridization, thus offering an alternative mechanism distinct from the DNA dense loading-enabled sharp transition scheme. The surface coverage of DNA on DNAAPSNSs is a key parameter dictating the kinetics of our DNA hybridization assay system. A decrease of the loading of DNA on DNA-APSNSs (DNA-APSNSs-S-L) to 64 (Supporting Information Table S4) results in the delayed onset of threshold turbidity at 8 min (Supporting Information Figure S13). The favorable contribution of the electrostatic interaction to the DNA hybridization kinetics is validated with several key 3503

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observations: (1) A high-efficiency neutralization of positive charge, by the reaction of DNA-APSNS surface amine with phenyl isothiocyanate (PITC) (ζ potential = ∼0 mV, Supporting Information Table S5), results in the emergence of threshold turbidity, as identified visually, at 5 min (Supporting Information Figure S14); (2) The development of threshold turbidity could be further delayed to 10 min if one switches the DNA-APSNS surface from protonated amino groups to negatively charged environment (ζ potential = −45.7 mV, Supporting Information Table S5) through the adsorption of poly(sodium-p-styrenesulfonate) (PSS) (Supporting Information Figure S15); (3) The hybridization of DNA1-APSNSs and HS-DNA2-AuNPs (Supporting Information Table S1)9,54 with target DNA3 exhibits a much slower hybridization kinetics with the advent of characteristic turbidity signature at ∼2 h (Supporting Information Figure S16); (4) A detailed measurement on the apparent hybridization kinetics of DNA-APSNSs (kH = 9.6 × 103 M−1·s−1, Supporting Information Table S7) and DNA-conjugated carboxylated PSNSs (∼197 nm, Supporting Information Figures S17 and S18, Table S8)55 (DNACPSNSs) (kH = 3.4 × 103 M−1·s−1, Supporting Information Table S9) indicates that the negatively charged environment is indeed unfavorable for the DNA binding event, generating a ∼3-fold decrease in the rate constant. Thermal desorption analysis indicates the likely existence of 31% noncovalently bound DNA besides covalently linked structure (Supporting Information Table S4). However, an APSNS assay system fabricated with electrostatic interaction-based noncovalent adsorption45 of nonaminated DNA provides a slower hybridization kinetics (kH = 2.5 × 103 M−1·s−1, Supporting Information Table S10), confirming that our threshold turbidity visualization scheme is facilitated by the covalent conjugation between aminated DNA and APSNSs. The appearance of threshold turbidity is, as expected, target concentration dependent, with the characteristic signature recognizable at 3, 15, and 25 min for 100, 10, and 5 nM of DNA3, respectively (Figure 4). Because of the expeditious development of threshold turbidity, the onset time can be determined accurately. Significantly, the difference in the threshold turbidity onset time could be used as a convenient handle for the quantification of target concentrations (Figure 4E, Supporting Information Figures S19−S22), with a perfect linear correlation (5 nM to 1 μM) if presented on a double logarithmic scale. In contrast, the thermodynamic end point analysis can not provide any observable or measurable signature for such a broad dynamic range. In fact, a precipitated state is invariably reached after 12 h of hybridization, and extinction spectroscopy only allows the differentiation of an extremely narrow range of target concentrations because of the largely plateaued signal readout (Supporting Information Table S11 and Figure S23). The visually distinct threshold onset time could also be translated to a kinetically definable time with a threshold extinction value as monitored by a UV−vis extinction spectrometer (Figure 5 and Supporting Information Figure S22). The detection limit with the current threshold turbidity visualization format is 2 nM (Supporting Information Figure S24), which could be further pushed down to 500 pM (Supporting Information Table S11 and Figure S23) if one exploits the end point analysis by the measurement of extinction spectroscopy. In principle, the threshold turbidity onset time could be shortened through the employment of an elevated concentration of DNA-APSNSs. Indeed, with the use

Figure 4. Macroscopic transmittance images of 100 μL PBSTW solutions of DNA1-APSNSs (0.376 nM) and DNA2-APSNSs (0.376 nM) after incubation in the presence (far left onward: 1 μM, 100 nM, 10 nM, and 5 nM) and absence (far right) of DNA3 for 0 (A), 3 (B), 15 (C), and 25 min (D). The threshold turbidity for 100 nM, 10 nM, and 5 nM was identified to be 3, 15, and 25 min, respectively. A linear correlation (E) between the threshold turbidity onset time and target concentration could be observed if the graph is plotted on a double logarithmic scale. Quantification of DNA concentration with the threshold turbidity onset time. A broad dynamic range (1 μM, 100 nM, 10 nM, and 5 nM) is warranted by the linear relationship on a double logarithmic scale.

of 8 × concentrated DNA-APSNSs, the advent of signature is decreased to ∼7 min for a target concentration of 10 nM (Supporting Information Figure S25), corresponding to a 2-

Figure 5. Characterization of DNA hybridization-driven assembly process for DNA-APSNSs by measuring the time-dependent extinction at 450 nm. Target concentration: 1 μM (filled square), 100 nM (hollow circle), 10 nM (hollow triangle), and 5 nM (hollow rhombus). The dashed red line denotes the approximate threshold extinction required for the visual identification of threshold turbidity. 3504

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fold rate enhancement in the assembly process. The size of DNA-APSNSs is not a key factor dictating the threshold onset time, as long as it falls in the right ready-for-transition regime (130−500 nm), which is demonstrated by the fairly effective signal generation process for DNA-APSNSs of ∼353 nm in diameter (Supporting Information Figure S1B) (∼2 min for 1 μM target) (Supporting Information Figure S26). The ability to differentiate single base mismatches4,56,57 has been the hallmark of the selectivity associated with a DNA assay system. Conventional diagnostic formats rely on the difference in thermodynamic stability of the DNA duplex to perform end point distinction of DNA with varied sequences. However, for certain DNA sequences, the end point differentiation can become extremely challenging due to the diminutive difference in the hybridization capacity. For example, under particular conditions, the melting transition of our assay system is vastly similar for a perfect target DNA3 and a mismatched SBM4-DNA3 (Supporting Information Table S1) with the single base mutation at the probe sequence middle of DNA1-APSNSs (melting temperature = 35 °C, fwhm = 1.6 °C, Supporting Information Figure S27). The challenging differentiation demand can be facilely met by the choice of an experimental setting (low salt concentration, 0.06 M NaCl) that permits the kinetic visual resolution of threshold turbidity (Supporting Information Figures S28A-F and S30). In fact, the threshold turbidity can be visualized at different times for different mismatched sequences (Supporting Information Table S1), including single base deletion (SBM1-DNA3), single base mismatch at probe head (SBM2-DNA3), single base insertion (SBM3-DNA3), and single base mismatch at probe middle (SBM4-DNA3). Importantly, the onset time can be employed as an operable parameter for the indexing of single base mismatch characteristics (Supporting Information Figures S28−S31). It should be noted that the type of base at the mismatch position is also a determinant of the onset time for threshold turbidity (SBM5-DNA3, SBM6-DNA3, and SBM7DNA3) (Supporting Information Table S1, Figures S29 and S31). To further prove the usefulness of the method, PCR products (double-stranded DNA, Supporting Information Table S1) containing part of sequence identical to DNA3 (PCR-DNA3) and single base mismatched strands (PCRSBM1-DNA3, PCR-SBM2-DNA3, PCR-SBM3-DNA3, and PCR-SBM4-DNA3) were subjected to the assay. Again, the onset time for threshold turbidity associated with various PCR products could be used for the identification of mismatch features (Supporting Information Figures S32 and S33).

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Article

ASSOCIATED CONTENT

S Supporting Information *

Materials and methods, surface functional group and DNA quantification, and DNA hybridization kinetics. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.Z. acknowledges support from the National Natural Science Foundation of China (20974044, 90923006) and the National Basic Research Program of China (2011CB935801).

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CONCULSION

In summary, a nanosphere-based ultrafast kinetic threshold turbidity visualization system has been developed for the assay of DNA hybridization. The diagnostic strategy allows for both target concentration quantification and differentiation of single base mismatches through registry of the threshold turbidity onset time. The positively charged assay environment on nanospheres not only contributes to the acceleration of signal generation but also establishes a likely framework for the creation of cooperative DNA binding architecture. The kinetic visual protocol complements conventionally used thermodynamic schemes and should provide an inspiring vision for the circumvention of assay issues when the analysis of ill-defined thermodynamic end points is impossible. 3505

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Analytical Chemistry

Article

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