DNA Recognition

Ultrasensitive and Closed-Tube Colorimetric LoopMediated Isothermal Amplification Assay Using Carboxyl-Modified Gold Nanoparticles Jacky K. F. Wong, Shea Ping Yip, and Thomas M. H. Lee* The detection of specific nucleic acid sequence in a simple manner can add great value to point-of-care diagnostics and on-site pathogen testing.[1] Gold nanoparticles (AuNPs), with their unique colorimetric property, are well-suited to this task.[2] AuNPs exhibit a characteristic surface plasmon resonance (SPR) absorption band in the visible light region and the exact spectrum is dependent on the interparticle distance. Specifically, particle aggregation gives rise to a red shift of the SPR absorption band and a concomitant red-to-purple color change. This property has been utilized for solution-phase colorimetric detection of specific nucleic acid sequences.[3–6] Nevertheless, these previously reported detection platforms did not possess all the essential attributes needed for practical applications, including high sensitivity, simple temperature control, no carryover contamination, and low cost. The vast majority of these platforms made use of oligonucleotide-modified AuNPs.[3a–d,4a,b,5a–g,6] One crucial aspect of effecting a visible color change is that the concentration of target sequence must be at nanomolar level.[3a–d] For real applications, a significantly higher sensitivity is required. Efforts have thus been devoted to coupling AuNP-based colorimetric detection with target amplification.[4a,b,5a–g,6] In this regard, isothermal techniques,[5a–g] which involve simple temperature control, are more desirable than thermal cycling techniques.[4a,b,6] Another major concern is that, for all isothermal[5] and most thermal cycling[4] (with three exceptions[6]) amplification-assisted platforms, post-amplification open-tube addition of AuNP probes (both oligonucleotidemodified and unmodified) was unavoidable, which poses a high risk of carryover contamination. The incompatibility of AuNP probes with amplification reactions is due to the thermal[6b,7] and/or dithiothreitol-induced[8] desorption of oligonucleotides from AuNPs followed by non-specific Dr. J. K. F. Wong, Dr. T. M. H. Lee Interdisciplinary Division of Biomedical Engineering The Hong Kong Polytechnic University Hung Hom, Kowloon, Hong Kong, China E-mail: [email protected] Prof. S. P. Yip Department of Health Technology and Informatics The Hong Kong Polytechnic University Hung Hom, Kowloon, Hong Kong, China DOI: 10.1002/smll.201302348 small 2014, 10, No. 8, 1495–1499

adsorption of enzyme onto AuNP surface (or just the latter part for unmodified AuNPs[6b,9]). Among the three closed-tube platforms (all were based on thermal cycling and oligonucleotide-modified AuNPs), the two with practically useful sensitivity required special preparation of oligonucleotide-modified AuNPs (silica coating[6b] or trithiolated oligonucleotide[6c]). Yet another issue with oligonucleotide-modified AuNPs is the high cost of thiol-modified oligonucleotides. Herein, we report a new closed-tube platform using loopmediated isothermal amplification (LAMP)[10] and 11-mercaptoundecanoic acid-modified AuNPs (MUA–AuNPs) that possesses all the ideal features for decentralized testing. The advantages of LAMP include versatility (applicable to DNA as well as RNA in single- and double-stranded forms),[10e] robustness (partially processed or unprocessed sample),[11] and ease of reagent transport and storage (lyophilized reagent).[11] Another useful feature of LAMP is the generation of pyrophosphate ion (P2O74−) as a reaction by-product, which forms the basis of turbidimetric[10c] or fluorescence[10d] monitoring in a closed-tube format. Hupp and co-workers pioneered the use of MUA–AuNPs for the sensing of divalent heavy metal ions (e.g., lead, cadmium, and mercury) based on ion-templated chelation.[12] They also showed that the aggregated particles could be redispersed by the addition of a strong metal ion chelator (ethylenediaminetetraacetic acid, EDTA). We hypothesize that magnesium ion (Mg2+), which plays an indispensable role in LAMP reaction as an enzyme cofactor, can trigger aggregation of MUA–AuNPs and P2O74− can then lead to deaggregation. In this work, the first task was to investigate the effects of Mg2+ and P2O74− on the solution color of MUA–AuNPs. AuNPs with a mean diameter of 15 nm were synthesized by citrate reduction method[6b,13] and functionalized with carboxyl groups by simple incubation with MUA (see the Supporting Information). The chelation between the carboxyl groups and Mg2+ is schematically illustrated in Figure 1a. The resulting aggregated MUA–AuNPs can be redispersed by the addition of P2O74−, which extracts the chelated Mg2+. To mimic the LAMP reaction condition, the aggregation and deaggregation experiments were performed at 65 °C. Upon the addition of Mg2+ (2 mM) to MUA–AuNPs (6 nM), the solution color changed immediately from red to purple. After standing for 1 h, dark purple precipitates were observed and the supernatant became clear (Figure S1a in the Supporting

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lambda DNA) appeared red (Figure 2b and Figure S2). Besides, agarose gel electrophoresis analysis of the reaction products showed that MUA–AuNPs did not have inhibitory effect on the LAMP reaction (Figure 2c). This is because AuNPs with closely packed self-assembled monolayer[8b,9b] (MUA in our case) would be more compatible with amplification reaction than oligonucleotidemodified[6b,8b] and unmodified[9] AuNPs. To our knowledge, this is the first demonstration of a AuNP-based colorimetric detection platform coupled to isothermal amplification in closed-tube format. Moreover, another advantage of MUA–AuNPs is their low cost as compared to oligonucleotide-modified AuNPs. The color difference between negative and positive samples was attained via a systematic optimization of the concentrations of Mg2+ and dNTPs. There are two standard combinations, one with 4 mM Mg2+ plus 0.4 mM each dNTP[10a,b] and the other one with 8 mM Mg2+ plus 1.4 mM each dNTP,[10d] but both did not Figure 1. Aggregation of 11-mercaptoundecanoic acid-modified gold nanoparticles (MUA– permit the purple-to-red color change AuNPs) by magnesium ion (Mg2+)-templated chelation process and reversible deaggregation for the positive sample (Figure S3a). In by pyrophosphate ion (P2O74−). a) Schematic showing the reversible aggregation and the former case, the maximum amount of 2+ 4− 2+ deaggregation of MUA–AuNPs by Mg and P2O7 , respectively. b) Upon the addition of Mg 4− (2 mM), the color of the MUA–AuNPs solution (particle concentration of 6 nM) turned from P2O7 that can be produced is 1.6 mM, 4− which can bind with 3.2 mM Mg2+. This red to purple. Subsequently, with the addition of P2O7 (1.4 mM) followed by incubation at 65 °C for 1 h, dark red precipitates were observed (Figure S1a in the Supporting Information). reduces the concentration of free Mg2+ to After mild sonication for ∼10 s, a red solution was obtained. a level (0.8 mM) at which MUA–AuNPs are still aggregated (Figure S4). In the Information). On the other hand, when the addition of Mg2+ latter case, although the theoretical maximum amount of genwas followed by P2O74− (1.4 mM), dark red precipitates were erated P2O74− (5.6 mM) can bind all Mg2+, only a small fracobserved (Figure S1a). With mild sonication, the precipitates tion is indeed produced (∼1.5 mM[10c] as dNTPs are in excess. were dispersed to give purple and red solutions, respectively To confirm this, a follow-up experiment was conducted with (Figure 1b). Of note, the deaggregation by P2O74− is only par- extra P2O74− (5.6 mM) added to the positive sample after the tial as evidenced by the more intense red color with EDTA LAMP reaction. With further incubation at 65 °C for 1 h and (a stronger Mg2+ chelator, Figure S1b). Another prerequisite then mild sonication, a red solution was observed (Figure S5). for colorimetric LAMP is that all other reaction components From the above analysis, and given that MUA–AuNPs were must not contribute to the deaggregation process, in par- in monodispersed state at 0.4 mM Mg2+ (Figure S4), we ticular deoxynucleoside triphosphates (dNTPs, the precur- tackled these issues by lowering the concentrations of Mg2+ sors of P2O74−). As shown in Figure S1b, dNTPs (0.35 mM and dNTPs (while keeping their ratios as the standard cases). each, i.e., 1.4 mM in total) had no observable effect on the Among the two new combinations (2 mM Mg2+ with 0.2 or 0.35 mM each dNTP), only the one with more dNTPs gave aggregated MUA–AuNPs. The second task was to demonstrate a proof-of-concept rise to the desired color change despite similar agarose gel experiment for the closed-tube colorimetric LAMP assay. electrophoresis results (Figure S3b). The third task was to evaluate the specificity of the colMUA–AuNPs were included in the reaction mixture along with the standard LAMP components. As depicted in the pro- orimetric LAMP assay. pBR322 DNA was used as the nonposed detection scheme (Figure 2a), MUA–AuNPs remain specific analyte. As expected, the sample containing pBR322 aggregated throughout the LAMP reaction in the absence of (105 copies) appeared purple whereas the sample containing a target sequence, while the initially aggregated particles are lambda DNA and pBR322 (105 copies each) appeared red gradually dispersed by the generated P2O74− in the presence (Figure 3a). The fourth task was to determine the detecof the target. The experimental results were consistent with tion limit. Figure 3b and Figure S6a show that samples with our expectation that a negative sample (without the target, 200 copies or more of the target DNA were visually distinthe model analyte used in this study was lambda DNA) guishable from the negative control (without the target). This appeared purple while a positive sample (with 105 copies of was further supported by a blue shift of the SPR absorption

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Ultrasensitive and Closed-Tube Colorimetric Loop-Mediated Isothermal Amplification Assay

Figure 2. MUA–AuNPs for closed-tube colorimetric loop-mediated isothermal amplification (LAMP) assay. a) Schematic illustration of the detection principle. MUA–AuNPs aggregate in Mg2+-containing LAMP reaction mixture. In the absence of target DNA sequence, the solution remains purple, whereas in the presence of target DNA sequence, LAMP produces P2O74− which extracts the chelated Mg2+ from the aggregated MUA–AuNPs and thus the solution turns red. b) Photograph of samples without (−ve) and with (+ve) the target sequence (105 copies of lambda DNA) subjected to LAMP for 1 h, followed by mild sonication for ∼10 s. c) Agarose gel electrophoresis of the LAMP reaction products. Lane M: 100 bp DNA ladder; lanes 1 and 2: controls without MUA–AuNPs; lanes 3 and 4: samples with MUA–AuNPs; lanes 1 and 3: samples without the target; and lanes 2 and 4: samples with the target.

band for the samples with 200 copies or more of the target DNA with reference to the negative control (Figure 3c and Figure S6b). The detection limit of our platform is the same as previously reported open-tube LAMP platforms[5f–h] and 3–6 orders of magnitude lower than that of other open-tube isothermal platforms.[5a–e] Agarose gel electrophoresis results indicate that MUA–AuNPs would not compromise the specificity and sensitivity of LAMP (Figure S7 and S8). The final task was to demonstrate the feasibility of running the colorimetric LAMP assay with temperature control by exothermic chemical reaction (iron oxidation in disposable air-activated hand warmer). The reaction tubes were put inside a paper cup containing a mixture of fresh and used contents of hand warmer in a mass ratio of ∼1:5 (Figure S9a; the temperature can be maintained at 60–65 °C for 1 h). A silicone oil overlay was added to the reaction mixture to prevent evaporation, which may significantly alter the concentrations of reactants (especially Mg2+) and thus affect the color change. Importantly, similar results were obtained with both the hand warmer- and equipment-based colorimetric LAMP assays (Figure S9b and Figure 2b). In conclusion, we have developed a novel closed-tube colorimetric LAMP assay using MUA–AuNPs. Our new platform enjoys all the advantages of high specificity and sensitivity, simple temperature control, visual colorimetric readout, worry-free carryover contamination control, short analysis time, and low cost. In particular, our method, with small 2014, 10, No. 8, 1495–1499

a detection limit of 200 copies per 20 µL reaction (17 aM), is among the most sensitive in all the reported AuNP-based colorimetric DNA detection methods (whether closed- or opentube; isothermal or thermal cycling amplification), which were typically in picomolar or femtomolar levels. Strikingly, LAMP has performance superior to other amplification techniques in terms of versatility, robustness, and ease of reagent transport and storage. Taken together, this technology has great potential for nucleic acid testing in decentralized settings as well as in resource-constrained laboratories. We will explore the possibility of real-time absorbance measurement using a small-sized and battery-operated colorimeter, thereby enabling accurate quantification of target copy number.

Experimental Section Aggregation and Deaggregation Tests for MUA–AuNPs: For aggregation test, a mixture containing MUA–AuNPs (6 nM) and 1× isothermal amplification buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% Tween 20, pH 8.8; New England Biolabs) was prepared. For deaggregation test, after 1 min incubation, the above mixture was supplemented with K4P2O7 (1.4 mM) or EDTA (2.8 mM) or dNTPs (0.35 mM each). These solutions were incubated at 65 °C for 1 h (GeneAmp PCR System 9700, Applied Biosystems). Colorimetric results were recorded before and after mild sonication for 10 s (WiseClean WUC-A01H ultrasonic cleaner,

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recorded before and after mild sonication for 10 s. Ultraviolet–visible spectra of the sonicated reaction products (70 µL scale) were measured using an Ultrospec 2100 pro UV/ visible spectrophotometer (GE Healthcare). For agarose gel electrophoresis analysis, the reaction products (8 µL product plus gel loading buffer) were run on a 2% gel in TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.0) at 120 V for 1.5 h. Then, the gel was stained with ethidium bromide (0.5 µg mL−1) for 10 min and visualized by UV transillumination. In the experiment with temperature control by exothermic chemical reaction, the reaction mixture was overlaid with silicone oil (10 µL). The reaction tube was put into a paper cup filled with hand warmer contents (17.5 g fresh contents were mixed with 82.5 g used contents; Nukupon, Kokubo) and incubated for 75 min as it took 15 min to reach the optimum LAMP reaction temperature of 60–65 °C.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Figure 3. Specificity and sensitivity of the colorimetric LAMP assay with MUA–AuNPs. a) Photograph of four samples with different templates (specific template of lambda DNA and non-specific template of pBR322 DNA): (from left to right) control sample without template; sample with lambda DNA; sample with pBR322 DNA; and sample with lambda DNA and pBR322 DNA. The amount of each template was 105 copies per reaction. b) Photograph of samples containing different amounts of the target sequence (0, 101, 102, 103, 104, and 105 copies of lambda DNA per reaction). c) Ultraviolet–visible spectra of the samples in b).

Daihan Scientific). For Mg2+ concentration-dependent aggregation test, different amounts of MgSO4 (0, 0.4, 0.8, and 1.2 mM) were added to MUA–AuNPs (6 nM). These solutions were incubated at room temperature for 10 min. Colorimetric results were recorded after mild sonication for 10 s. Colorimetric LAMP Assay: Six primers were used for amplifying lambda DNA[10b] (HPLC-purified, Integrated DNA Technologies), including FIP: 5′-CAGCCAGCCGCAGCACGTTCGCTCATAGGAGATATGGTAGAGCCGC-3′; BIP: 5′-GAGAGAATTTGTACCACCTCCCACCGGGCACATAGCAGTCCTAGGGACAGT-3′; F3: 5′-GGCTTGGCTCTGCTAACACGTT-3′; B3: 5′-GGACGTTTGTAATGTCCGCTCC-3′; loop F: 5′-CTGCATACGACGTGTCT-3′; and loop B: 5′-ACCATCTATGACTGTACGCC-3′. The reaction mixture (20 µL) comprised 1× isothermal amplification buffer, FIP (0.8 µM), BIP (0.8 µM), F3 (0.2 µM), B3 (0.2 µM), loop F (0.4 µM), loop B (0.4 µM), dNTPs, betaine (1 M), Bst 2.0 DNA polymerase (0.32 units µL−1, New England Biolabs), specific template of lambda DNA (or no template control or non-specific template of pBR322 DNA), and MUA–AuNPs (6 nM). The concentrations of MgSO4 and dNTPs were 2 mM and 0.35 mM (each dNTP), respectively, unless otherwise stated. LAMP was carried out at 65 °C for 1 h (GeneAmp PCR System 9700). Colorimetric results were

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Acknowledgements This work was supported by The Hong Kong Polytechnic University (project code: A-PL44).

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Received: August 1, 2013 Revised: November 6, 2013 Published online: March 13, 2014

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Ultrasensitive and closed-tube colorimetric loop-mediated isothermal amplification assay using carboxyl-modified gold nanoparticles.

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