Fluorescence and Visual Detection of Single Nucleotide

ARTICLE pubs.acs.org/Langmuir

Fluorescence and Visual Detection of Single Nucleotide Polymorphism Using Cationic Conjugated Polyelectrolyte Yifan Wang,† Ruoyu Zhan,† Tianhu Li,‡ Kan-Yi Pu,† Yanyan Wang,† Yoke Cheng Tan,§ and Bin Liu*,† †

Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, Singapore 117567, Singapore ‡ Division of Chemistry and Biological Chemistry, Nanyang Technology University, Singapore 637371, Singapore § DSO National Laboratories, Singapore 117510, Singapore ABSTRACT: We report a simple assay for visual detection of single nucleotide polymorphisms (SNPs) with good sensitivity and selectivity. The selectivity is determined by Escherichia coli (E. coli) DNA ligase mediated circular formation upon recognition of the point mutation on DNA targets. Rolling cycle amplification (RCA) of the perfect-matched DNA target is then initiated using the in situ formed circular template in the presence of Phi29 enzyme. Due to amplification of the DNA target, the RCA product has a tandem-repeated sequence, which is significantly longer than that for the SNP strand. Direct addition of a cationic conjugated polymer of poly[9,90 -bis(60 -(N,N,N-trimethylammonium)hexyl)fluorene-co-9,90 -bis(2-(2-(2-(N,N,N-trimethylammonium)ethoxyl)-ethoxy)ethyl)fluorene tetrabromide] containing 20 mol% 2,1,3-benzothiadiazole (PFBT20) into the RCA solution leads to blue-whitish fluorescent color for SNP strand and yellowish fluorescent color for amplified DNA, due to PFBT20/DNA complexation induced intrachain/interchain energy transfer. To further improve the contrast for visual detection, FAM-labeled peptide nucleic acid (PNA) was hybridized to each amplified sequence, which is followed by the addition of poly{2,7-[9,9-bis(60 -N,N,N-trimethylammoniumhexyl)]fluorene-co2,5-difluoro-1,4-phenylene dibromide} (PFP). The PNA/DNA hybridization brings PFP and FAM-PNA into close proximity for energy transfer, and the solution fluorescent color appears green in the presence of target DNA with a detection limit of 1 nM, which is significantly improved as compared to that for most reported visual SNP assay.

S

ingle nucleotide polymorphisms (SNPs) are the most abundant forms of genetic variations, occurring every few hundred to few thousand base pairs in genomic DNA.1
3 In recent years, there has been increasing demand to develop simple, rapid and cost-effective bioassays for SNP detection since it is associated with numerous genetic and hereditary diseases.4,5 Conventional methods for SNP detection heavily rely on time-consuming gel electrophoresis for fragment analysis of endonuclease cleavage.6
8 The recently developed techniques include oligonucleotide ligation,9
11 primer extension,12
14 allele-specific DNA hybridization,15 or electrochemical typing.16,17 These methods provide accurate validation but need expensive instruments performed in modern laboratories. Simplicity, low cost, and rapidity with high accuracy are essential for diagnosis in disaster situations or in poorly equipped rural areas. In this regard, visual detection has distinctive advantages as it eliminates the need for an analytical instrument. So far, the absorbance change of organic dyes and distance-dependent optical properties of aggregated gold nanoparticles have been frequently used to develop visual assays for SNP detection.18
22 These assays generally require several DNA probes or modified DNA under stringent temperature control and low salt concentration. It is highly desirable to develop room temperature label-free assays for rapid and sensitive detection of SNP under mild conditions. r 2011 American Chemical Society

Cationic conjugated polymers (CCPs) are a new generation of materials that have been used for naked-eye sensing of biomolecules.23
33 The conformational change of cationic polythiophene was reported to show different color response to single-stranded DNA (ss-DNA) and double-stranded DNA (ds-DNA).23,24 In addition, in our previous studies, we have shown that cationic polyfluorene derivatives containing 5
30 mol % substitution of 2,1,3-benzothiadiazole (BT) units could facilitate target detection and quantification with multiple color emission.25
32 Complex formation between these polymers and negatively charged biomolecules gives rise to polymer aggregation, which increases the local concentration of BT units, leading to enhanced BT emission. These assays largely rely on nonspecific electrostatic interactions, which have good response to pure analytes in solution but generally show poor selectivity for analytes with small variations. Recent studies have shown that rolling cycle amplification (RCA) is a simple and elegant technique to generate long ssDNA molecules with tandem repeats under isothermal conditions.34,35 Received: September 22, 2011 Revised: November 2, 2011 Published: November 02, 2011 889

dx.doi.org/10.1021/la203714e | Langmuir 2012, 28, 889–895

Langmuir

ARTICLE

RCA has been widely used in sensitive detection of DNA and other targets including protein and small molecules through the use of DNA aptamer and allosteric DNAzymes in solution or on microarrays.36
43 The high sensitivity and specificity of RCA based assay is due to the stringent strand matching requirement for ligation and its high amplification efficiency.44 To perform RCA, a circular template needs to be synthesized first from a padlock probe in which the 50 - and 30 -termini hybridize precisely to the target DNA and are joined by a DNA ligase.44 After isothermal amplification of the circular template, the RCA product generally contains thousands of repeated sequences. The general strategies to quantify RCA products require hybridization with fluorescently labeled complementary signal probes, followed by signal collection using microarray scanner for assays on solid support (e.g., microsphere), or using confocal microscope/flow cytometry or electrophoresis to count the individual probes in solution.37,38,45
47 These approaches generally require labeled probes and sophisticated instruments. Recently, RCA has been reported to combine with gold nanoparticle aggregates for single nucleotide polymorphisms detection.48 Although this strategy relies less on an instrument, it requires the RCA products to be digested by a restricting endonuclease, so that the cleaved DNA fragments can mediate the aggregation of gold nanoparticle-tagged DNA probes. It is highly desirable that a simple instrument-free signal reporting system could be developed for RCA assays. In this contribution, we report a simple visual SNP detection strategy by taking advantage of the optical response of CCP and target hybridization induced DNA ligation followed by rollingcircle amplification. Both DNA amplification and detection are conducted at room temperature in the absence of any specialized instrument. Two strategies have been demonstrated to show how the CCP could be used with RCA assays for sequence specific DNA detection with high sensitivity and good selectivity. The manuscript is organized as follows. We start with the optimization of RCA conditions, which is followed by CCP based visual detection and quantification of SNP. The results reveal that incorporation of RCA into CCP based assays can significantly improve the visual detection sensitivity. The assay performance has been further improved using PNA-FAM as the probe for SNP detection with improved visual contrast. The combination of CCP with RCA-based detection method allows SNP detection in just a few hours without a sophisticated instrument, which is of high importance in emergency situations or in poorly equipped battle field or rural areas.

The HPLC-purified 6-carboxyfluorescein (FAM) labeled PNA (FAM-OOTGA AGG TTG T-Lys-Lys) was purchased from PANAGENE, Korea. Poly[9,90 -bis(60 -(N,N,N-trimethylammonium)hexyl)fluorene-co-9,90 -bis (2-(2-(2-(N,N,N-trimethylammonium)ethoxyl)-ethoxy)-ethyl)fluorene tetrabromide] containing 20 mol% 2,1,3-benzothiadiazole (PFBT20) and poly{2,7-[9,9-bis(60 -N,N,N-trimethylammoniumhexyl)]fluorene-co2,5-difluoro-1,4-phenylene dibromide} (PFP) were synthesized according to the literature.30 The fresh stock solutions (1 mM) for both polymers were prepared based on repeat units (RU). The molecular weight of PFBT20 and PFP repeat units were determined to be 1065.7 g/mol and 720.6 g/mol, respectively. All the PL experiments were carried out at 24 °C. Milli-Q water (18.2 MΩ) was used for all experiments. Padlock Probe Hybridization. 100 nM padlock DNA (DNAp) and different concentrations (0
100 nM) of target DNA (DNAWT) or SNP strand (DNASNP) were mixed together in 1 E. coli. DNA ligase buffer (30 mM Tris-HCl, 4 mM MgCl2, 1 mM dithiothreitol (DTT), 26 μM NAD+, 50 μg/mL BSA, pH 8.0). The mixture was further diluted to 100 μL using Milli-Q water. The mixture was then heated to 80 °C for 5 min in an incubator, slowly cooled down to room temperature, and stored at 4 °C for another 2 h before further use. Circulization of Padlock Probe. After hybridization between the padlock probe (100 nM) and target DNA or SNP strand at a specific concentration (0
100 nM) in 1 E. coli ligase buffer, 10 U of E. coli DNA ligase (1 μL, 10 U/μL) was subsequently added into 40 μL of each hybridization mixture. The mixtures were then kept at room temperature for 30 min. The ligation reactions were terminated by heating up the mixture to 65 °C for 20 min. Rolling Circle Amplification. A 20 μL portion of the above ligation reaction mixture was mixed with 20 μL of 1 RCA reaction buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM (NH4)2SO4, and 4 mM DTT to a final volume of 40 μL. 1 mM dNTPs (dATP, dTTP, dCTP, and dGTP, New England Biolab), 0.1 mg/mL BSA, and 20 U phi29 DNA polymerase (2 μL, 10 U/μL) were subsequently added, and the mixture was incubated at room temperature for 30 min. The amplification was terminated by heating up to 65 °C for 10 min.

Optimization of Rolling Circle Amplification Reaction Time. For the optimization of RCA reaction time, E. coli DNA ligase

(1 μL, 10 U/ μL) was added into 40 μL of 10 nM hybridized padlock probe and reacted for 30 min. After heating inactivation at 65 °C for 20 min, the RCA reaction was performed at room temperature for 2, 5, 10, 30, and 60 min before termination. 10 μL of each RCA product was transferred separately to a new tube and was heated up to 65 °C for 10 min. The obtained RCA products were analyzed using 0.8% agarose gel electrophoresis (100 V for 2 h) followed by ethidium bromide staining. Optimization of PFBT20. To optimize the amount of PFBT20 for each reaction, 10 μL of 100 nM RCA reaction solution was diluted to 1 mL using Milli-Q water. Different amounts of PFBT20 (10
4 M) were added dropwise at 1 μL intervals into the reaction solution, and the emission spectra were collected in the range 380
700 nm upon excitation at 365 nm. Visual Detection of SNP using PFBT20. 10 μL of 100 nM RCA reaction solution was mixed with 1 μL of PFBT20 (10
3 M) polymer solution and further diluted to 100 μL using Milli-Q water. The photos of fluorescent solutions were taken under a hand-held UV lamp with λmax = 365 nm. To quantify the fluorescence signal, the solutions for both target and SNP strand were further diluted to 1 mL, and the emission spectra were collected in the range 380
700 nm upon excitation at 365 nm. Visual detection of various concentrations (10
6, 10
7, 10
8, 10
9, and 10
10 M) of target and SNP strands was performed following the same procedure. SNP Analysis in the Absence of RCA. 100 nM of padlock probe and 100 nM of target DNA or SNP strand were hybridized in 1 E. coli DNA ligase buffer for 4 h. 10 U of E. coli DNA ligase (1 μL, 10 U/μL) was subsequently added. The mixtures were then kept at room temperature for 30 min. The reactions were terminated by heating up

’ EXPERIMENTAL SECTION Instrumentation. Fluorescence was measured using a PerkinElmer LS 55 equipped with a xenon lamp excitation source and a Hamamatsu (Japan) 928 PMT, using 90° angle detection for solution samples. For energy transfer experiments, the excitation beam intensity has been automatically corrected with a correction file provided by the instrument supplier. Photographs of the polymer solutions were taken using a Canon EOS 400D digital camera under a hand-held UV lamp with λmax = 365 nm. Oligonucleotides and Chemicals. All oligonucleotides (DNAWT, 5 0 AAA TAC AAC CTT CAA ATA AAA 3 0 ; DNASNP , 5 0 AAA TAC AAC CTA CAA ATA AAA 3 0 ; DNAp, 5 0 p-AGG TTG TAT TTC ATC AGA ACT CAC CTG TTA GAC GCC ACC AGC TCC AAC TGT GAA GAT CGC TAT ATG ATG GTT TTA TTT GA 30 ) were purchased from first BASE Pte Ltd., Singapore. E. coli DNA ligase and phi29 DNA polymerase were obtained from New England Biolabs Inc. 890

dx.doi.org/10.1021/la203714e |Langmuir 2012, 28, 889–895

Langmuir

ARTICLE

Figure 1. (A) Schematic illustration of PFBT20-assisted detection. (B) The chemical structure of PFBT20. (C) Normalized absorption and PL spectra of PFBT20 at 1 μM in water upon excitation at 365 nm. to 65 °C for 20 min. 10 μL of each ligation mixture was diluted to 1 mL of PFBT20 (1 μM) polymer solution, and the emission spectrum was collected in the range 380
700 nm upon excitation of PFBT20 at 365 nm. Visual Detection of SNP Based on PNA-FAM. 40 μL of RCA reaction solution (10 nM) was hybridized with 0.4 μL of FAM-labeled PNA (100 μM), and the mixture was heated up to 65 °C for 5 min and cooled down to room temperature in 2 h. 10 μL of the reaction solution was diluted to 100 μL using Milli-Q water and was further mixed with 1 μL of PFP (10
4 M) polymer solution. The photos of fluorescent solutions were taken under a hand-held UV lamp with λmax = 365 nm. To quantify the fluorescence signal, 10 μL of the reaction solution was diluted to 1 mL using Milli-Q water. 1 μL of PFP (10
4 M) polymer solution was added into the reaction solution, and the emission spectra were collected from 380 to 700 nm upon excitation at 365 nm. Visual detection of SNP at various concentrations (10
7, 10
8, 10
9, and 10
10 M) using PNA-FAM was performed following the same procedure.

addition, Bacillus anthrax can directly cause acute fatal disease anthrax via inhalation transmission and is considered as a bioterror agent, while B. cereus is simply a ubiquitous soil bacterium.50 An 80-mer oligonucleotide (DNAp) was designed to be partially complementary to DNAWT so that the 30 - and 50 -terminal sequence of the 80-mer could be hybridized to the target. The 21-mer target fragment then acts as a “splint” template which allows circulization of the 80-mer padlock probe in the presence of DNA ligase, when the base at 30 -end of the 80mer is perfectly matched to the target. Subsequently, DNAWT switches its role to be the primer which initiates rolling-circle amplification to generate long single-stranded DNA with tandem repeats. Addition of PFBT20 to the DNA solution leads to complex formation between PFBT20 and the long DNA molecules, which induces the polymer aggregation and changes the solution fluorescent color to yellow. On the other hand, since the SNP strand has a single base mismatch at the ligation site, it will lead to failures in the circular formation process, and the subsequent RCA reaction will not be initiated. Further addition of PFBT20 into this solution will not induce obvious emission changes, as the polymerization will stop right after an 80-mer dsDNA is formed and the solution fluorescent color remains bluish. Optical Properties of the PFBT20. PFBT20 (structure shown in Figure 1) was synthesized according to our previous report.30 The polymer has a molecular weight of 11 000 and a polydispersity of 3.0. The absorption and emission spectra of PFBT20

’ RESULTS AND DISCUSSION Assay Principle. The CCP-assisted SNP detection strategy is shown in Figure 1. Two 21-mer fragments of B. anthracis A0248 (DNAWT) and B. cereus G9842 (DNASNP) were selected as the target and the SNP sequence, as the original DNA sequences were found to be identical for a continuous stretch of more than 1000 bases and differed only by one single nucleotide.49 In 891

dx.doi.org/10.1021/la203714e |Langmuir 2012, 28, 889–895

Langmuir

ARTICLE

Figure 2. Normalized fluorescence intensity of 10 nM ssDNA generated by RCA with reaction time varying from 2 to 60 min. The inset shows the gel picture of RCA products after gel electrophoresis (100 V for 2 h) on 0.8% agarose, followed by ethidium bromide stain. Reaction times for lanes 1
4 are 2, 5, 10, and 30 min, respectively.

Figure 4. Normalized PL spectra of PFBT20 (purple), control solution (green), and 10 μL each of 100 nM [DNAWT] assay (red) and 100 nM [DNASNP] assay (black) in the presence of 1 μM PFBT20 upon excitation at 365 nm. Inset shows the photos of the fluorescence from the corresponding solutions under a hand-held UV lamp with λmax = 365 nm.

with water to 1 mL before PFBT20 was added. As shown in Figure 3, upon drop-by-drop addition of PFBT20, there is an increase in both emission bands at 415 and 580 nm, respectively. The band centered at 415 nm corresponds to the emission from the fluorene segments, while the peak at 580 nm is due to the BT emission. With the polymer addition, complexation between PFBT20 and negatively charged biomolecules in solution induces polymer aggregation, giving rise to enhanced energy transfer from the fluorene segments to the BT units, and the I580nm/415nm is significantly higher than that for the pure polymer emission as shown in Figure 1. Upon excitation at 365 nm, the highest ratio for I580nm/415nm is obtained at [PFBT20] = 1.0 μM, which gives the largest fluorescence difference relative to that for the pure polymer, and is thus used as the optimized PFBT20 concentration for SNP detection. Specificity Study of PFBT20-Based Visual SNP Detection. To evaluate the assay selectivity, two RCA products (10 μL of 100 nM RCA reaction solution diluted in 1 mL water) for the target and the SNP sequence together with a control have been prepared. 1 μL of 1 mM PFBT20 was subsequently added to each sample, and the normalized PL spectra upon excitation at 365 nm are shown in Figure 4. The highest intensity at 580 nm is observed for the solution containing the target sequence (red curve in Figure 4). This is due to the fact that rolling circle amplified anthrax target possesses long ssDNA, which has a large amount of negative charges. These negative charges are able to effectively induce polymer aggregation and generate strong BT emission. When the solution is visualized under a hand-held UV lamp, it appears yellow (tube 1 in Figure 4). On the other hand, the SNP solution has shown a much weaker BT emission (black curve in Figure 4). As the template circulate formation is difficult in the presence of SNP, the presence of the 80-bp template is not able to initiate significant polymer aggregation. This leads to a bluish fluorescent solution (tube 2 in Figure 4). The control solution in the absence of DNA also gives some BT emission signal as PFBT20 also responds to the ligase, polymerase, and ions used for the enzymatic reactions (green curve in Figure 4), which serves as the background signal in this study. Nevertheless, the PL intensity and solution fluorescent color difference is sufficient for SNP detection. In addition, other control experiments by addition of the same amount of polymer to DNA ligation product alone in the absence of enzyme phi29 could not yield any detectable difference for both wide type DNA and SNP

Figure 3. PL spectra of 100 nM RCA product upon addition of PFBT20 at 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, and 2.5 μM upon excitation at 365 nm.

are shown in Figure 1. PFBT20 has an absorption maximum at 375 nm, and its emission shows a maximum at 415 nm with almost no obvious emission band in the 500
650 nm region. In dilute solution, the polymer emission color is blue. Optimization of Rolling Circle Amplification Reaction. The hybridization between the 80-mer and target was conducted following a standard hybridization protocol. As the effect of various conditions on ligation has been reported in the literature,17 in this study, we only focus on the optimization of time required for RCA reaction. In the first ligation step, 10 nM of DNAp was hybridized with an equal amount of DNAWT followed by addition of 10 U of E. coli DNA ligase and reacted for 30 min to form the circular structure. The ligation reaction was stopped by deactivation at 65 °C for 10 min. The formation of circular structure has been confirmed by monitoring the UV absorption at 254 nm after S1 nuclease digestion and gel electrophoresis. To optimize the time required for RCA, a series of reactions were performed for 2
60 min on the basis of the same concentration of the circular structure and phi29 polymerase. The obtained RCA products were analyzed through 0.8% agarose gel and stained with ethidium bromide. Figure 2 shows the changes of PL intensity in the major gel band with respect to the amplification time. The PL intensity increases sharply from 2 to 30 min and further increasing the reaction time does not lead to a significant increase in the band intensity. This could be due to the fact that the RCA reaction has reached equilibrium or the enzyme phi29 polymerase has significantly lost its reactivity after 30 min. These results indicate that 30 min is ideal for the RCA reaction in this study. Optimization of PFBT20 Concentration. Upon completion of the RCA reaction, 10 μL of the reaction solution was diluted 892

dx.doi.org/10.1021/la203714e |Langmuir 2012, 28, 889–895

Langmuir

ARTICLE

Figure 5. (A) Photographs of solutions for DNAWT and DNASNP assays using E. coli DNA ligase at different DNA concentrations ranging from 10
7 to 10
11 M after addition of 1 μM PFBT20. (B) Changes of the normalized solution photoluminescence intensity at 580 nm with increased initial concentrations of DNA used for RCA reaction.

Figure 6. (A) Schematic illustration of FRET based detection. (B) The chemical structure of PFP used in this study.

sequence. As a result, it is the DNA ligation followed by rolling-circle amplification that is able to provide the DNA detection selectivity. Visual DNA Quantification with PFBT20. To test the assay feasibility for visual DNA quantification, various concentrations (10
6, 10
7, 10
8, 10
9, and 10
10 M) of DNAWT and DNASNP were used for ligation and RCA amplification under optimized conditions as discussed above. As shown in Figure 5, in the presence of 1 μM PFBT20, there is a clear fluorescent color difference between the target DNA and SNP at any concentration that is above 10
9 M. The target solutions appear yellowish upon addition of PFBT20 while SNP solutions stay blue-whitish. The difference in fluorescent color exists even when the concentration is decreased to 10
10 M. In addition to visual detection, fluorescence spectroscopy study reveals that the normalized fluorescence intensity at 580 nm

for the RCA solution containing the perfect cDNA increases with the initial DNA concentration used for RCA reaction, which indicates that the polymer based assay is indeed suitable for DNA quantification with a limit of detection of 30 pM based on 3σ from three independent measurements. FRET-Based Visual SNP Detection. Although we have successfully achieved visual SNP strand detection with 1 nM sensitivity using PFBT20, the solution fluorescent color varies for SNP solutions at different concentrations, which is due to nonspecific interaction between PFBT20 and the SNP strands together with the reaction medium. To minimize fluorescent signal variation for the SNP assay and to further enhance the color contrast, we replace PFBT20 with a FRET pair of PFP and a FAM-labeled PNA probe into the system (Figure 6). The detection and quantification of targets take advantage of PNA 893

dx.doi.org/10.1021/la203714e |Langmuir 2012, 28, 889–895

Langmuir

ARTICLE

Figure 7. Absorption spectra of PFP (black) and FAM (green) and the emission spectrum of PFP (red) in 10 mM PBS buffer (pH = 7.4).

Figure 9. Photographs of different FRET assays for DNAWT and DNASNP in the presence of PFP. E. coli DNA ligase is employed, and the DNA concentration ranges from 10
7 to 10
10 M.

the cDNA after RCA, and the solution has shown green fluorescence upon excitation with a hand-held UV lamp (tube 1 in Figure 8). On the other hand, the SNP solution remains fluorescent blue due to the absence of long single-stranded RCA product which is complementary to PNA probe (tube 2 in Figure 8). The red curve corresponds to the solution containing the anthrax target DNA, while the SNP solution has shown much weaker emission intensity at 540 nm. Sensitivity of FRET-Based Visual DNA Detection. To evaluate whether the FRET based fluorescence assay is able to provide DNA quantification, the RCA reaction solutions (10
7, 10
8, 10
9, and 10
10 M for initial target and SNP sequence concentrations) were hybridized with 1 μM of FAM-labeled PNA for 2 h. After addition of 1 μL of PFP (10
4 M) solution, the samples were collected and placed under a hand-held UV lamp for visualization. As shown in Figure 9, there is a very clear fluorescent color difference between the target DNA and SNP at a concentration as low as 1 nM. The target solutions appear green upon addition of PFP while the SNP solutions stay blue. The color contrast is further improved as compared to that shown in Figure 5A.

Figure 8. Normalized PL spectra of solutions containing 10 nM target (red) and 10 nM SNP (black) after RCA upon addition of 1 μM FAMPNA, followed by addition of 1 μL of 10
4 M PFP upon excitation at 365 nm. Inset shows the corresponding solution fluorescent color under a hand-held UV lamp with λmax = 365 nm.

hybridization induced energy transfer between PFP and PNA/RCA product duplexes. The good overlap between the emission spectrum of PFP and the absorption spectrum of FAM (Figure 7) indicates possible energy transfer between them, which has been demonstrated in our previous study.51 The short PNA probe is designed to be complementary to the tandem repeats of ssDNA formed after rolling-circle amplification. As PNA probe is neutral, there is no electrostatic interaction between the PNA and PFP. Upon hybridization with the RCA product, the PNA and tandem DNA would form negatively charged complexes that would bring the polymer and FAM into close proximity for energy transfer. On the other hand, when ligation and RCA are not successful in the presence of SNP, the 80-mer mediated energy transfer is weak, so that the obtained signal would be significantly different from that for the cDNA. We choose a PNA probe52 instead of a DNA probe for hybridization to the RCA product so as to avoid the interaction between the probe and PFP so that the RCA product could be directly visualized and quantified without complicated instrument or separation steps. As compared to PFBT20-based assay, the effect of enzymes and buffer ions on polymer emission is less reflected in the background signal as the fluorescence mainly comes from polymer amplified FAM emission. To demonstrate the assay selectivity, two RCA products (10 μL of 100 nM diluted to 1 mL water) for the target and the SNP sequence have been prepared. After hybridization with 1 μM PNA-FAM at room temperature for 2 h, 1 μL of PFP (10
4 M)53 has been subsequently added to each sample and the normalized PL spectra upon excitation at 365 nm are shown in Figure 8. Efficient energy transfer is observed for the solution containing

’ CONCLUSION We report a simple assay for naked-eye SNP detection, which takes advantage of enzymatic amplification and the optical response of water-soluble cationic conjugate polymers to achieve high detection sensitivity and selectivity without the needs of any specialized instrument for both DNA amplification and detection. As compared to gold-nanoparticle-based naked-eye detection, this assay eliminates the need for temperature control and also does not require multiple probes or DNA modification. As DNA quantification in traditional RCA assays requires hybridization with fluorescently labeled complementary signal probes, followed by signal collection using microarray scanner for assay on solid support (e.g., microsphere), or using confocal microscope/flow cytometry or electrophoresis to count the individual probes in solution, direct stain of the RCA products with a cationic conjugated polyelectrolyte or using a dye-labeled peptide nucleic acid probe allows naked-eye DNA quantification in the absence of any complicated instrument, which simplifies the RCA based DNA assay process. The developed simple and portable assay is of practical importance in emergency situations or in poorly equipped battle field or rural areas. 894

dx.doi.org/10.1021/la203714e |Langmuir 2012, 28, 889–895

Langmuir

ARTICLE

’ AUTHOR INFORMATION

(26) Zhan, R. Y.; Fang, Z.; Liu, B. Anal. Chem. 2010, 82 (4), 1326–1333. (27) Liu, B.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126 (7), 1942–1943. (28) Hong, J. W.; Henme, W. L.; Keller, G. E.; Rinke, M. T.; Bazan, G. C. Adv. Mater. 2006, 18 (7), 878–882. (29) Pu, K. Y.; Liu, B. Macromolecules 2008, 41 (18), 6636–6640. (30) Shi, J. B.; Pu, K. Y.; Zhan, R. Y.; Liu, B. Macromol. Chem. Phys. 2009, 210 (15), 1195–1200. (31) Yu, D. Y.; Zhang, Y.; Liu, B. Macromolecules 2008, 41 (11), 4003–4011. (32) Pu, K. Y.; Cai, L. P.; Liu, B. Macromolecules 2009, 42 (16), 5933–5940. (33) Pu, K. Y.; Zhan, R. Y.; Liu, B. Chem. Commun. 2010, 46 (9), 1470–1472. (34) Lizardi, P. M.; Huang, X. H.; Zhu, Z. R.; Bray-Ward, P.; Thomas, D. C.; Ward, D. C. Nat. Genet. 1998, 19 (3), 225–232. (35) Hardenbol, P.; Baner, J.; Jain, M.; Nilsson, M.; Namsaraev, E. A.; Karlin-Neumann, G. A.; Fakhrai-Rad, H.; Ronaghi, M.; Willis, T. D.; Landegren, U.; Davis, R. W. Nat. Biotechnol. 2003, 21 (6), 673–678. (36) Fire, A.; Xu, S. Q. Proc. Natl. Acad. Sci. U.S.A. 1995, 92 (10), 4641–4645. (37) Li, J.; Zhong, W. W. Anal. Chem. 2007, 79 (23), 9030–9038. (38) Konry, T.; Hayman, R. B.; Walt, D. R. Anal. Chem. 2009, 81 (14), 5777–5782. (39) Jarvius, J.; Melin, J.; Goransson, J.; Stenberg, J.; Fredriksson, S.; Gonzalez-Rey, C.; Bertilsson, S.; Nilsson, M. Nat. Methods 2006, 3 (9), 725–727. (40) Zhang, S. B.; Wu, Z. S.; Shen, G. L.; Yu, R. Q. Biosens. Bioelectron. 2009, 24 (11), 3201–3207. (41) Schweitzer, B.; Wiltshire, S.; Lambert, J.; O’Malley, S.; Kukanskis, K.; Zhu, Z. R.; Kingsmore, S. F.; Lizardi, P. M.; Ward, D. C. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (18), 10113–10119. (42) Lee, H. J.; Wark, A. W.; Corn, R. M. Analyst 2008, 133 (8), 975–983. (43) Zhao, W. A.; Ali, M. M.; Brook, M. A.; Li, Y. F. Angew. Chem., Int. Ed. 2008, 47 (34), 6330–6337. (44) Stromberg, M.; Goransson, J.; Gunnarsson, K.; Nilsson, M.; Svedlindh, P.; Stromme, M. Nano Lett. 2008, 8 (3), 816–821. (45) Schweitzer, B.; Roberts, S.; Grimwade, B.; Shao, W. P.; Wang, M. J.; Fu, Q.; Shu, Q. P.; Laroche, I.; Zhou, Z. M.; Tchernev, V. T.; Christiansen, J.; Velleca, M.; Kingsmore, S. F. Nat. Biotechnol. 2002, 20 (4), 359–365. (46) Blab, G. A.; Schmidt, T.; Nilsson, M. Anal. Chem. 2004, 76 (2), 495–498. (47) Li, N.; Jablonowski, C.; Jin, H. L.; Zhong, W. W. Anal. Chem. 2009, 81 (12), 4906–4913. (48) Li, J. S.; Deng, T.; Chu, X.; Yang, R. H.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. Anal. Chem. 2010, 82 (7), 2811–2816. (49) Ash, C.; Farrow, J. A. E.; Dorsch, M.; Stackebrandt, E.; Collins, M. D. Int. J. Syst. Bacteriol. 1991, 41 (3), 343–346. (50) Radnedge, L.; Agron, P. G.; Hill, K. K.; Jackson, P. J.; Ticknor, L. O.; Keim, P.; Andersen, G. L. Appl. Environ. Microbiol. 2003, 69 (5), 2755–2764. (51) Liu, B.; Bazan, G. C. J. Am. Chem. Soc. 2006, 128 (4), 1188–1196. (52) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. Nature 1993, 365 (6446), 566–568. (53) Li, K.; Liu, B. Anal. Chem. 2009, 81 (10), 4099–4105.

Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT The authors are grateful to the National University of Singapore (R279-000-301-646), Ministry of Defense (R279-000-301-232), and The Temasek Defense Systems Institute (R279-000-305-592, R279-000-305-422 and R279-000-305-232) for financial support. R.Z. and K.-Y.Pu thank Singapore Ministry of Education (R-279000-255-112) for financial support. ’ REFERENCES (1) Irizarry, K.; Kustanovich, V.; Li, C.; Brown, N.; Nelson, S.; Wong, W.; Lee, C. J. Nat. Genet. 2000, 26 (2), 233–236. (2) McCarthy, J. J.; Hilfiker, R. Nat. Biotechnol. 2000, 18 (5), 505–508. (3) Kirk, B. W.; Feinsod, M.; Favis, R.; Kliman, R. M.; Barany, F. Nucleic. Acids. Res. 2002, 30 (15), 3295–3311. (4) Venter, J. C.; et al. Science 2001, 291 (5507), 1304–1351. (5) Halushka, M. K.; Fan, J. B.; Bentley, K.; Hsie, L.; Shen, N. P.; Weder, A.; Cooper, R.; Lipshutz, R.; Chakravarti, A. Nat. Genet. 1999, 22 (3), 239–247. (6) Hall, J. G.; Eis, P. S.; Law, S. M.; Reynaldo, L. P.; Prudent, J. R.; Marshall, D. J.; Allawi, H. T.; Mast, A. L.; Dahlberg, J. E.; Kwiatkowski, R. W.; de Arruda, M.; Neri, B. P.; Lyamichev, V. I. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (15), 8272–8277. (7) Ross, P.; Hall, L.; Smirnov, I.; Haff, L. Nat. Biotechnol. 1998, 16 (13), 1347–1351. (8) Schmalzing, D.; Belenky, A.; Novotny, M. A.; Koutny, L.; SalasSolano, O.; EI-Difrawy, S.; Adourian, A.; Matsudaira, P.; Ehrlich, D. Nucleic Acids. Res. 2000, 28 (19), e43. (9) Landegren, U.; Kaiser, R.; Sanders, J.; Hood, L. Science 1988, 241 (4869), 1077–1080. (10) Wu, D. Y.; Wallace, R. B. Genomics 1989, 4 (4), 560–569. (11) Tobe, V. O.; Taylor, S. L.; Nickerson, D. A. Nucleic Acids. Res. 1996, 24 (19), 3728–3732. (12) Huh, Y. S.; Lowe, A. J.; Strickland, A. D.; Batt, C. A.; Erickson, D. J. Am. Chem. Soc. 2009, 131 (6), 2208–2213. (13) Xu, Y. Z.; Karalkar, N. B.; Kool, E. T. Nat. Biotechnol. 2001, 19 (2), 148–152. (14) Sando, S.; Abe, H.; Kool, E. T. J. Am. Chem. Soc. 2004, 126 (4), 1081–1087. (15) Wallace, R. B.; Shaffer, J.; Murphy, R. F.; Bonner, J.; Hirose, T.; Itakura, K. Nucleic Acids. Res. 1979, 6 (11), 3543–3557. (16) Liu, G.; Wan, Y.; Gau, V.; Zhang, J.; Wang, L. H.; Song, S. P.; Fan, C. H. J. Am. Chem. Soc. 2008, 130 (21), 6820–6825. (17) Huang, Y.; Zhang, Y. L.; Xu, X. M.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. J. Am. Chem. Soc. 2009, 131 (7), 2478–2480. (18) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105 (4), 1547–1562. (19) Li, H. X.; Rothberg, L. J. J. Am. Chem. Soc. 2004, 126 (35), 10958–10961. (20) Xu, W.; Xue, X. J.; Li, T. H.; Zeng, H. Q.; Liu, X. G. Angew. Chem., Int. Ed. 2009, 48 (37), 6849–6852. (21) Li, J. S.; Deng, T.; Chu, X.; Yang, R. H.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. Anal. Chem. 2010, 82 (7), 2811–2816. (22) Komiyama, M.; Ye, S.; Liang, X. G.; Yamamoto, Y.; Tomita, T.; Zhou, J. M.; Aburatani, H. J. Am. Chem. Soc. 2003, 125 (13), 3758–3762. (23) Ho, H. A.; Boissinot, M.; Bergeron, M. G.; Corbeil, G.; Dore, K.; Boudreau, D.; Leclerc, M. Angew. Chem., Int. Ed. 2002, 41 (9), 1548–1551. (24) Nilsson, K. P. R.; Inganas, O. Nat. Mater. 2003, 2 (6), 419–424. (25) Tang, Y. L.; Teng, F.; Yu, M. H.; An, L. L.; He, F.; Wang, S.; Li, Y. L.; Zhu, D. B.; Bazan, G. C. Adv. Mater. 2008, 20 (4), 703–705. 895

dx.doi.org/10.1021/la203714e |Langmuir 2012, 28, 889–895