Ultrasensitive Detection of Single Nucleotide Polymorphism in Human

Jan 26, 2015 - Ultrasensitive Detection of Single Nucleotide Polymorphism in Human Mitochondrial DNA Utilizing Ion-Mediated Cascade Surface-Enhanced ...
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Ultrasensitive Detection of Single Nucleotide Polymorphism in Human Mitochondrial DNA Utilizing Ion-Mediated Cascade SurfaceEnhanced Raman Spectroscopy Amplification Muling Shi,†,§ Jing Zheng,†,§ Yongjun Tan,† Guixiang Tan,† Jishan Li,† Yinhui Li,† Xia Li,⊥ Zhiguang Zhou,⊥ and Ronghua Yang*,†,‡ †

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha, 410082, China ‡ School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha, 410004, China ⊥ Xiangya Second Hospital of Central South University, Changsha, 410082, China S Supporting Information *

ABSTRACT: Although surface-enhanced Raman spectroscopy (SERS) has been featured by high sensitivity, additional signal enhancement is still necessary for trace amount of biomolecules detection. In this paper, a SERS amplified approach, featuring “ions-mediated cascade amplification (IMCA)”, was proposed by utilizing the dissolved silver ions (Ag+) from silver nanoparticles (AgNPs). We found that using Ag+ as linkage agent can effectively control the gaps between neighboring 4-aminobenzenethiol (4-ABT) encoded gold nanoparticles (AuNPs@4-ABT) to form “hot spots” and thus produce SERS signal output, in which the SERS intensity was proportional to the concentration of Ag+. Inspired by this finding, the IMCA was utilized for ultrasensitive detection of single nucleotide polymorphism in human mitochondrial DNA (16189T → C). Combining with the DNA ligase reaction, each target DNA binding event could successfully cause one AgNP introduction. By detecting the dissolved Ag+ from AgNPs using IMCA, low to 3.0 × 10−5 fm/μL targeted DNA can be detected, which corresponds to extractions from 200 nL cell suspension containing carcinoma pancreatic β-cell lines from diabetes patients. This IMCA approach is expected to be a universal strategy for ultrasensitive detection of analytes and supply valuable information for biomedical research and clinical early diagnosis.

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of numerous Raman-reporter-encoded nanoparticles,14−16 in this case, enhancement of Raman signal occurs when the reporter molecule resides in the proper gap between the neighboring nanoparticles.17 Though various attempts have been explored, these strategies still depend on the direct conversion of target recognition events into signal readouts merely in one single step, which remains one of the bottlenecks for further improving the sensitivity of SERS sensors. The challenge can be addressed by employing amplification intermediates, through which multiple steps magnified signal outputs can be produced from one target recognition event. Intermediary ions triggered colorimetric,18 electrochemical,19−21 mass spectrometry,22 and fluorescence detection23,24 have been developed, whereas a SERS-based method has not been reported previously. Inspired by this, we developed an ion-mediated cascade SERS amplification technique (IMCA) to achieve ultrasensitive biomolecule

he quantitative and sequence-specific analysis for nucleic acid with extremely low abundance plays essential roles in diagnosis and treatment of genetic diseases. Surface-enhanced Raman spectroscopy (SERS) stands out as a significant analytical technique with high sensitivity and molecular specificity.1,2 Nanoparticle-based SERS detection of nucleic acid and biomolecules has recently received considerable attention.3−10 This includes utilizing metal colloids, such as gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs), to enhance local electromagnetic field by forming “hot spots” to produce SERS signal output. However, further sensitivity improvements still remains an enormous challenge. Focusing on this, researchers have advanced additional enhancement strategies to endow SERS sensors with a lower limit of detection (LOD). One design type, for example, is designed to achieve high SERS signal enhancement through exquisite fabrication of SERS-active substrates,11 such as periodic nanoarray on metal films,12,13 whereas it requires exhausting and expensive preparation of specialized material. Another additional enhancement strategies aims at amplifying the signal output by target or nucleic acid enzyme-mediated aggregation © XXXX American Chemical Society

Received: October 26, 2014 Accepted: January 26, 2015

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Figure 1. Schematic illustrations of IMCA SERS detection of SNP. (A) AuNPs@4-ABT was adopted as a SERS-active detection substrate which undergoes aggregation introduced by Ag+. (B) SERS detection of mtDNA T16189C mutation of type 2 diabetes via ligase reaction.

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EXPERIMENTAL SECTION Materials and Instruments. All DNA oligonucleotides (Table S1, Supporting Information) were synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China). Streptavidin-coated silica microbeads (SiMBs, nonporous, 5 μm) were purchased from Bangs Laboratories, Inc. and dispersed at 0.1 mg/mL in 100 mM phosphate-buffered saline (PBS, pH 7.4). E. coli DNA ligase with 10× ligation reaction buffer was obtained from Takara Biotechnology Co., Ltd. (Dalian, China). 4-Aminobenzenethiol (4-ABT) was purchased from Alfa Aesar. All other chemicals were of analytical reagent grade and were used as received, unless otherwise stated. Ultrapure water obtained from a Millipore water purification system (18 MΩ, Milli-Q, Millipore) and nonstick tubes (DNA loBind tube, Eppendorf, German) were used in all assays. SERS measurements were performed using a confocal microprobe Raman instrument (Ram Lab-010, Horiba Jobin Yvon, France), and spectra were acquired at 25 °C using a 632.8 nm He−Ne laser and a 50× long working objective lens (8 mm). For each Raman spectrum, the laser output power was 3 mW, the diameter of laser focus point was 2 μm, the collecting time was 20 s, and the width of the slit and the size of the pinhole were set as 100 μm and 1000 μm, respectively. The AuNPs and AgNPs used in the experiment were characterized by UV−vis absorption spectra on a Hitachi U-4100 UV−vis spectrophotometer (Kyoto, Japan) and transmission electron microscopy using a JEOL-3010 microscope. Construction of IMCA Sensing Platform. AuNPs were prepared according to previous literature.26 Briefly, AuNPs (the average diameter, 60 ± 8 nm) were prepared through a seeded growth method by reduction of HAuCl4 with hydroxylamine hydrochloride on citrate-reduced AuNPs seeds (∼30 nm). The resulting gold colloids were diluted with ultrapure water by 1:1

detection in this paper. As proof of concept, a single recognition event would introduce one AgNP which could be dissolved into numerous silver ions (Ag+) with oxidant in our design. The releasing numerous Ag+ then acted as intermediates to induce 4-aminobenzenethiol (4-ABT) encoded AuNPs (AuNPs@4-ABT) aggregation and thus achieved IMCA. Through such an amplification method, even a trace amount of target can bring about obvious signal readouts. Moreover, by selecting an appropriate recognition element, this strategy can act as a universal sensing platform to realize various analytes detection. As further practical application, we applied this IMCA strategy for the detection of single nucleotide polymorphisms (SNPs) in human mitochondrial DNA by taking advantage of the fidelity of DNA ligase. The integrity of mitochondrial DNA (mtDNA) is crucial for maintaining normal cellular oxidative phosphorylation while mutations of mtDNA have been reported to be a cause of maternally inherited diabetes mellitus.25 Since the content of mtDNA in cell is relative low, the reliable methods with high sensitivity for mutations in mtDNA are thus highly desired, especially in early clinical diagnosis of mitochondria-related hereditary diseases, such as diabetes. In our study, the mutant and wild type of mtDNA containing the SNP site (16189T → C) were chosen as targets. The result demonstrated that our constructed IMCA can lead to an extremely low LOD for mtDNA which corresponds to extractions from 200 nL of cell suspension containing about 120 carcinoma pancreatic β-cells from diabetes patients. To the best of our knowledge, this work demonstrated for the first time one to realize the application of IMCA in bioanalysis, which provides a promising platform for genetic target analysis and clinic biomedical application. B

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Analytical Chemistry volume ratio (3.3 × 10−8 M) followed by the incubation with 4ABT solution (1.0 × 10−5 M) with alcohol−water mixed solvent for 5 min. For each assay, 20 μL sample solution containing Ag+ was added into 500 μL AuNP@ 4-ABT solution with phosphate buffer (PBS) at pH 7.0, and the SERS spectrum was collected after incubation in a sealed tube in darkness for 10 min. SNP Detection. The biotin-labeled P0 was immobilized on streptavidin-coated SiMBs as the capture probe following the procedure of our previous work.16 The mixtures of P0 and SiMBs were vortexed at room temperature for 1 h, followed by washing three times with PBS using centrifugation to remove excessive P0 that did not conjugate to SiMBs. The conjugates were dispersed in PBS and stored at 4 °C at a concentration of 0.2 mg/mL. Prior to binding, T1 and T2 DNA were heated to 95 °C in solution followed by rapid cooling in an ice bath for uncoiling. Then the P1 was added and the ligation reaction was conducted according to our previous work.27 To differentially denature the duplex formed by T1 or T2, the solutions were heated to 43 °C for 20 min, which was identified by thermally melting. Then the solutions were centrifuged at 1000 rpm for 3 min and the precipitate was collected. The AgNPs were prepared based on the combination of the seed-mediated growth and the Lee−Meisel method by thermal reduction of AgNO3 with citrate according to the previous literature.28 Then the AgNPs were purified by gradient centrifugation and screened to obtain particles with uniform size (average size of 70 nm) before adding to the precipitate and reacted at room temperature for 1 h. Then, they were washed three times with PBS using centrifugation at 1000 rpm to remove free AgNPs. Afterward, 30% hydrogen peroxide (H2O2) solution was used to dissolve AgNPs retained on SiMBs to yield free Ag+. Finally, the supernatant was collected and mixed with buffer as sample solution and then incubated with AuNP@4-ABT. Application in Real Sample. Carcinoma pancreatic β-cell lines were obtained from The Second Xiangya Hospital of Central South University (Hunan, China). Cells were cultured on a 75 cm2 cell culture flask in dulbecco’s modified eagle medium (DMEM) with 10% fetal bovine serum, 584 mg L−1 Lglutamine, 100 μg mL−1 streptomycin, and 100 units mL−1 penicillin in 5% CO2 atmosphere at 37 °C. Cells were detached from the substrate with 0.25% trypsin in media followed by centrifugation. The supernatant was subsequently discarded and replaced by 10 mL of media. The cell suspension was then passed through a 40 μm sterile cell strainer and the cells were counted with a hemocytometer. The mitochondria were extracted using a Tissue Mitochondria Isolation Kit (Beyotime, Shanghai) according to the manufacture’s protocol followed by mitochondrial DNA extraction using a DNA Mini Preparation Kit with Spin Column (Beyotime, Shanghai), then the PCR products of mitochondrial DNA were applied to our approach.

mediated method. To facilitate the following 4-ABT encoding and minimize the preoccupation of the surface encoding site by ions from the reducing agent, hydroxylamine hydrochloride instead of commonly used sodium citrate was adopted as a reducing agent. This is mainly due to the fact that the concentration of introduced chloridion during the process of hydroxylamine-reduced AuNPs synthesis is lower than citrate anions from citrate-reduce AuNPs.29 As characterized by transmission electron microscopy (TEM), AuNPs were shown to be uniform and monodispersed with an average size of 60 ± 8 nm and the UV−vis absorption spectrum shows that λmax was 552 nm (Figure S1 in the Supporting Information). Then the AuNPs were incubated with 4-ABT to form AuNPs@4-ABT through the S−Au bond. To avoid the unexpected aggregation caused by the high concentration of 4ABT, the AuNPs were not fully covered with 4-ABT. In our experiment, 4-ABT and the diluted AuNPs were incubated with a molar ratio of 303:1 and the surface coverage of 4-ABT was then evaluated to be 2.97 pmol/cm2, assuming perfect adsorption probability and an occupation area of 22 Å2.30 Next, Ag+ was introduced and the aggregation of AuNPs@4ABT was investigated. Aggregation of AuNPs@4-ABT mediated by Ag+ was first evidenced by the TEM image in Figure 2A. Upon Ag+ addition, TEM images demonstrated

Figure 2. (A) TEM images of AuNPs@4-ABT upon the addition of 1 μM Ag+. Inset: TEM images of AuNPs@4-ABT without Ag+ addition. (B) UV−visible absorption of AuNPs@4-ABT before (blue trace) and after (pink trace) the addition of 1 μM Ag+. Inset: photograph showing the corresponding color change of AuNPs@4-ABT before (a) and after (b) the addition of 1 μM Ag+.

obvious aggregation of AuNPs@4-ABT compared with that without Ag+ addition as shown in the inset. The neighboring particles keep the certain gap ∼1 nm (indicated with the red arrow in Figure 2A and calculated from the statistical result of a series of TEM images), which is denoted as a representative“hot-spot”. The corresponding UV−vis spectra of bare AuNPs@ 4-ABT and the Ag+-mediated AuNPs@4-ABT aggregation are shown in Figure 2B. From the result we can see that upon 1 μM Ag+ addition, a red shift of plasmon band was observed accompanied by a red-to-blue color change. Then X-ray photoelectron spectroscopy (XPS) and Fourier transform-infrared (FT-IR) experiments were carried out to further confirm the manner of interaction between Ag+ and the AuNP@4-ABT. The XPS sample of dried powders was obtained by centrifugation of AuNPs@4-ABT suspension to remove free Ag+. As shown in Figure S2A in the Supporting Information, in the XPS survey spectrum of Ag+-AuNPs@4ABT, two new peaks at 368 and 374 cm−1 of Ag appeared compared with AuNPs@4-ABT. The high-energy Ag 3d doublets in the Ag 3d spectrum (Figure S2B in the Supporting

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RESULTS AND DISCUSSION Characterization of IMCA Sensing Platform. The design of our IMCA technique mainly based on the finding that Ag+ could mediate the aggregation of AuNPs@4-ABT, as illustrated in Figure 1A. We discovered that the introduction of Ag+ to the AuNPs@4-ABT solution could cause AuNPs aggregation by forming N → Ag ← N coordination compounds, and an obvious SERS spectrum induced by the subsequent formed “hot spots” could be observed. First, to attain the ideal match of the excitation wavelength (632 nm) with the plasmon band, the large size of AuNPs were synthesized by the two-step seed C

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field, as seen in curve a. However, a stronger SERS signal was demonstrated upon Ag+ addition (curve b).The observed Raman bands centered at 1142, 1391, and 1440 cm−1 can be assigned to b2-modes of the 4-ABT molecule.34 The SERS enhancement, I/I0, of the most prominent Raman peak at 1142 cm−1 was estimated to be 52.3-fold by 1 μM Ag+. According to methods in the literature,4 an enhancement factor of ∼2.05 × 10 9 was determined (for details, see the Supporting Information), a factor ∼102 to 103 greater than typical average factor of colloidal SERS.35 In this case, ideal Raman signals enhancement occurred due to the effective electromagnetic coupling of the localized surface plasmon of AuNPs. Figure 3B reveals that the value of I/I0 increased upon increasing Ag+ concentration from 500 pM to 5 μM. In the range from 5.0 × 10−10 to 1.0 × 10−7 M, the value of I/I0 was a good linear fit to the concentration of Ag+, and the correlation coefficient (R) was estimated to 0.9928. When the Ag+ concentration increased to about 1.0 × 10−7 M, or even higher, the SERS signal remained fairly constant and a plateau was reached due to the interaction between Ag+ and AuNPs@4-ABT finally reaching the equilibrium state in terms of kinetics and thermodynamics. Therefore, it can be concluded that this IMCA actually provides a significant amplification of Raman signal intensity by several orders of magnitude. To confirm the selectivity of AuNPs@4-ABT to Ag+, the effect of a series of other metal ions was also explored (Figure S5 in the Supporting Information). The values of I/I0 were analyzed upon 2 μM Cu2+, Hg2+, Zn2+, Fe2+, Cd2+ addition, respectively, in place of Ag+ (1 μM). It can be seen that only Cu2+ and Hg2+ which can coordinate with the amine group aroused a slight signal increase while other ions induce a negligible signal change. The result shows that other ions cannot interact with 4-ABT as strong as Ag+; therefore, AuNPs@4-ABT should be a superior sensor for Ag+. It even showed good sensitivity when exposed to 20% newborn bovine serum (NBS), 2 μM DNA, or 0.2 mg/mL trypsin (Figure S6 in the Supporting Information), indicating its ability to tolerate interference from real biological samples. Detection of SNP Utilizing IMCA. The cytosine for thymidine substitution (T → C) at nucleotide position (np) 16189, which lies in the human mtDNA control region for replication and transcription, has been reported to be associated with insulin resistance in Asians.32,36,37 Therefore, the discrimination of 16189T → C polymorphism is of great significance to cast light on its association with type 2 diabetes and could be used as a reference for the prediction of the risk of type 2 diabetes in Asian people. In our design, two 24 bp oligonucleotide sequences originated from human mtDNA, T1 and T2, were chosen as mutant and wild type targets, respectively (sequence is shown in Table S1, see the Supporting Information). In the assay, as shown in Figure 1B, the capture probe P0 (sequences were shown in Table S1 in the Supporting Information) was employed and immobilized on the surface of streptavidin functionalized silica microbeads (SiMBs) through the streptavidin−biotin interaction. In the presence of T1, one terminus of T1 hybridized with P0 and another terminus can hybridize with the signal transduction probe P1 (sequences were shown in Table S1 in the Supporting Information). As for P1, the 3′-end was labeled with a sulfhydryl group for AgNPs functionalization while the other end was phosphorylated for DNA ligation. Since T1 is complementary to P0 and P1, the perfect duplex structure was formed upon P1 addition and the ligation reaction catalyzed by E. coli DNA ligase was carried

Information) were possibly attributed to the interaction of silver with the amino group which suggested the interaction of Ag+ with the amino group of 4-ABT.31 While in the FT-IR spectrum of Ag+-AuNPs@4-ABT (Figure S3 in the Supporting Information), the peak demonstrated at 1380 cm−1 could possibly be attributed to diammine complexes of Ag+,32 which further validated the formation of N → Ag+ ← N between 4ABT and Ag+ as proposed in Figure 1A. These results can demonstrate that Ag+ can be used as a linkage agent to control the gaps between neighboring AuNPs@4-ABT for producing “hot spots”, which holds the potential of significant amplification of Raman signal intensity by several orders of magnitude through electromagnetic field enhancement. SERS Enhancement of IMCA. To attain ideal SERS signal enhancement of our constructed IMCA, experiment conditions such as pH and incubation time were optimized. We first investigated the incubation time of 4-ABT and AuNPs during the process of 4-ABT encoding (Figure S4A in the Supporting Information). The SERS enhancement, I/I0, of the most prominent Raman peak at 1142 cm−1 was investigated, where I0 and I are the SERS intensities at 1142 cm−1 in the absence and presence of Ag+, respectively. The highest the value of I/I0 was achieved at 5 min. Therefore, 5 min was chose as the optimal incubation time for AuNPs modification to achieve the best response sensitivity. Since it has been reported that the SERS spectrum of 4-ABT is pH-dependent,33 we then investigated the value of I/I0 under different pH to obtain optimal detection performance (Figure S4B in the Supporting Information). In acidic solutions, 4-ABT was protonated which went against the interaction with Ag+. However, high background (I0) tended to debase the value of I/I0 in alkaline solutions. Therefore, the SERS signal enhancement of IMCA was performed at pH 7.0 in PBS buffer. The incubation time of AuNPs@4-ABT and Ag+ also affects the detection performance, as shown in Figure S4C in the Supporting Information, the value of I/I0 increased rapidly with the incubation time upon Ag+ addition and trended to attain a constant value at 10 min, indicating that the formed N → Ag+ ← N between 4-ABT and Ag+ has reached saturation. In our subsequent experiment, 10 min was selected as the optimal time for the Ag+-mediated AuNPs@4-ABT aggregation. Figure 3A displays a set of SERS spectra of AuNPs@4-ABT in solution obtained under optimal conditions. The AuNPs@4ABT shows obscure SERS signals by the weak surface plasmon

Figure 3. (A) SERS spectra of AuNPs@4-ABT before (black trace) and after (red trace) the addition of 1 μM Ag+ along with the peak assignment. (B) SERS intensity ratio, I/I0, of the 1142 cm−1 band, is plotted against the concentrations of Ag+. The inset shows a linear relationship between I/I0 and Ag+ concentration at 1.0 × 10−9 to 1.0 × 10−7 M. Error bars show the standard deviation of three experiments. The collection time for each spectrum was 15 s and the accumulation was set as 3. D

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the one-base mismatched target DNA T2 was added, the two short probes of P0 and P1 could not be linked by DNA ligase to form stable duplexes because of the extremely low ligation yield when mismatched sequences are located at positions close to the ligation junction. Under these circumstances, P1 were removed in the following washing step and AgNPs could not be conjugated on the surface of SiMB. Therefore, the AuNPs@4ABT could not be aggregated due to Ag+ lacking and very weak SERS signal was observed (curve c). To further demonstrate the SERS signal amplified by our IMCA approach, other method adopted AgNPs directly as SERS substrate were also investigated for control. The experiment was designed by direct employing 4-ABT@AgNPs as signal output. Herein, AgNPs were first encoded with 4-ABT and then conjugated with the sulfhydryl group of P1 using the Ag−S bond. In the presence of T1, one terminus of T1 hybridized with P0 and another terminus could serve as the ligation template. Upon P1 addition, the ligation reaction between P0 and P1 catalyzed by E. coli DNA ligase was carried out, and the formed long double strand DNA was stable under 43 °C. Then, 4-ABT@AgNPs were added and then conjugated with the sulfhydryl group of P1 to produce the SERS signal. As shown in curve d, due to the limited SERS enhancement of single 4-ABT@AgNP on the surface of SiMB, the addition of 1 nM T1 only resulted in weak SERS signal state. In comparison, the SERS signal upon addition of the same concentration of T1 for our IMCA approach is estimated to 30-fold enhancement than that of directly sensed by 4-ABT@AgNPs, demonstrating the superiority in effective signal enhancement of our proposed platform over other methods. We next measured the SERS signal improvement as functions of T1 concentration. Figure 5A illustrates the SERS

out. The resulted long double strand DNA is stable and can sustain harsh wash conditions under a certain temperature which was determined from melting analysis (about 43 °C, Figure S8; for details see the Supporting Information). AgNPs (70 ± 5 nm, representative SEM image is shown in Figure S7; see the Supporting Information) were then added subsequently and conjugated to the sulfhydryl group of P1 using the Ag−S bond. After centrifugation, the conjugated AgNPs caused by T1 addition were dissolved with H2O2 and the releasing Ag+ could mediate the aggregation of AuNPs@4-ABT. Thus, obvious SERS signal was demonstrated. As for the detection of T2 which contains one mismatched sequence at the position close to the ligation junction, the ligation was not successful due to the high fidelity of the DNA ligase to a perfect duplex structure and thus P1 was washed in the following washing step under the same temperature. Thus, no AgNPs could be conjugated to the surface of SiMB in the subsequent procedure. Since the number of Ag+ constituted one AgNP is estimated to be about 105 which is calculated according to the reported literature,38 one T1 hybridization would generate numerous dissolved Ag+ to effectively enhance the SERS signal which holds potential to possess satisfactory analytical performance even when the target DNA is at a trace concentration. First, the AgNPs conjugated and dissolved by H2O2 on the SiMBs surface in the presence of T1 or T2 with the aid of DNA ligation were confirmed by the scanning electron microscope (SEM). As shown in Figure S9 in the Supporting Information, upon T1 addition and then mediated by DNA ligation, SEM images showed that AgNPs were immobilized on the surface of SiMBs and many bright spots appeared. The corresponding result of energy-dispersive X-ray spectrometric analysis in the right panel shows the peak of silver on the surface of SiMB. After dissolving by H2O2, the vast majority of bright spots disappeared and the surface of SiMB were similar to bare ones. However, as for T2 addition, no aggregates appeared on the surface of SiMB after thermal treatment and thus indicated almost no AgNP was successfully conjugated. Therefore, these DNA ligation-mediated AgNPs-encoded SiMBs have the potential of providing numerous Ag+ for substantial signal amplification. Then, we measured the SERS spectra of our constructed IMCA nanoplatform before and after T1 addition. As shown in Figure 4A, no obvious SERS signals could be observed due to the weak surface plasmon field in the absence of T1 (curve a). However, upon addition of T1 to the mixture, a stronger SERS signal was demonstrated by IMCA (curve b). Whereas, when

Figure 5. (A) SERS spectra for different concentrations of target DNA T1 (0, 50 fM, 100 fM, 1 pM, 10 pM, 100 pM, and 1 nM). (B) Dependence of I/I0 on different concentrations of target DNA dilutions. The inset shows linear calibration of I/I0 vs T1 concentration. Error bars show the standard deviation of three experiments. The collection time for each spectrum was 15 s and the accumulation was set as 3.

intensity increases dramatically as functions of different concentrations of T1. Change of Raman intensity at 1142 cm−1 (9b) with and without T1 termed as I/I0 was quantitatively analyzed and showed a good linear fit to the concentration of T1 in the range from 1.0 × 10−11 to 1.0 × 10−13 M (Figure 5B). The LOD at 3σ is 30 fM, which corresponds to 3.0 × 10−5 fM DNA in 1 μL sample. The result is equivalent to the amount of mtDNA extracted from 200 nL of carcinoma pancreatic β-cell suspension, demonstrating its satisfactory sensitivity which holds potential to apply in real sample. To evaluate a SNP detection assay, specificity is also the most important measurement. The specificity of our proposed assay

Figure 4. SERS spectra obtained from Ag+-mediated cascade SERS amplification approach in the presence of buffer (curve a), 1 nM T1 (curve b) and 1 nM T2 (curve c). Curve d was obtained by employing 4-ABT-modified AgNPs directly as Raman tags in the presence of T1. E

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is in general determined by the fidelity of the DNA ligase to perfect duplex structures. Initially, we mixed the oligonucleotides of T2 and T1 at different mole ratios of 10 000:1, 5000:1, 1000:1, 499:1, 99:1, and 9:1 with a total concentration of 5 nM. The blank reaction contained 5 nM of the wild-type target T2 only. The results are displayed in Figure 6. We can see that the

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ASSOCIATED CONTENT

S Supporting Information *

More experimental details about synthesis of AuNPs and AgNPs and spectroscopic data as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 86-731-88822523. Fax: 86-731-88822523. E-mail: [email protected]. Author Contributions §

M. Shi and J. Zheng contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the financial support through the National Natural Science Foundation of China (Grants 21405038, 21135001, 21305036, 21475036, and J1103312), the Foundation for Innovative Research Groups of NSFC (Grant 21221003), the “973” National Key Basic Research Program (Grant 2011CB91100-0), and the Fundamental Research Funds for the Central Universities.

Figure 6. Selectivity of this IMCA SERS sensing platform. Histogram showing the capacity to measure the perfect-matched target in a mixture of T1 and T2. The mole ratios of T2 and T1 is 10000:1, 5000:1, 1000:1, 499:1, 99:1, 9:1, respectively, with a total concentration of 5 nM. The standard deviations obtained by three repeated measurements are shown as the error bars.

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intensity increment is linearly correlated to the concentration of the perfectly matched target T1. Furthermore, an obvious intensity increase still can be seen when only 500 fM T1 was added to the blank mixture (a wild type strands T2 to mutant T1 ratio of 10 000:1). This result well demonstrates the extraordinary capability of our assay in detecting a scarce mutation among a large quantity of wild types along with the high sensitivity and wide dynamic range. Furthermore, we performed this assay on crude extracts of carcinoma pancreatic β-cell lines from diabetes patients. The results of 10 cases coincided with the Sanger sequencing results completely (Figure S10 in the Supporting Information), demonstrating the applicability of this approach in clinical early diagnosis.

CONCLUSION

An IMCA approach with high sensitivity is proposed in this paper. One recognition event would produce one AgNP, which could be dissolved into numerous Ag+. The numerous releasing Ag+ then acted as intermediates to achieve AuNPs@4-ABT aggregation and thus enhanced SERS signals. The result demonstrated that the IMCA approach could effectively discriminated 16189T → C polymorphism in human mitochondrial DNA at trace concentration combining with the fidelity of DNA ligase, suggesting its capacity as another alternative for SNP detection. Most importantly, this strategy possesses excellent generality which can be used as a versatile SERS sensing platform and various analytes can be detected by altering corresponding recognition element. Moreover, multiorder assembly of the nanostructure such as hybridization chain reaction (HCR) or rolling-circle amplification (RCA) could be explored to enable our constructed platform to realize multiorder amplification for ultrasensitive detection in the further work. F

DOI: 10.1021/ac504000p Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/ac504000p Anal. Chem. XXXX, XXX, XXX−XXX