Biosensors and Bioelectronics 65 (2015) 191–197

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Sensitive detection of point mutation using exponential strand displacement amplification-based surface enhanced Raman spectroscopy Si-qiang Huang 1, Juan Hu 1, Guichi Zhu 1, Chun-yang Zhang n Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong 518055, China

art ic l e i nf o

a b s t r a c t

Article history: Received 30 June 2014 Received in revised form 29 September 2014 Accepted 13 October 2014 Available online 18 October 2014

Accurate identification of point mutation is particularly imperative in the field of biomedical research and clinical diagnosis. Here, we develop a sensitive and specific method for point mutation assay using exponential strand displacement amplification (SDA)-based surface enhanced Raman spectroscopy (SERS). In this method, a discriminating probe and a hairpin probe are designed to specifically recognize the sequence of human K-ras gene. In the presence of K-ras mutant target (C-T), the 3′-terminal of discriminating probe and the 5′-terminal of hairpin probe can be ligated to form a SDA template. Subsequently, the 3′-terminal of hairpin probe can function as a primer to initiate the SDA reaction, producing a large amount of triggers. The resultant triggers can further hybridize with the discriminating probes to initiate new rounds of SDA reaction, leading to an exponential amplification reaction. With the addition of capture probe-modified gold nanoparticles (AuNPs) and the Rox-labeled reporter probes, the amplified triggers can be assembled on the surface of AuNPs through the formation of sandwich hybrids of capture probe–trigger–reporter probe, generating a strong Raman signal. While in the presence of K-ras wild-type target (C), neither ligation nor SDA reaction can be initiated and no Raman signal is observed. The proposed method exhibits high sensitivity with a detection limit of 1.4 pM and can accurately discriminate as low as 1% variant frequency from the mixture of mutant target and wild-type target. Importantly, this method can be further applied to analyze the mutant target in the spiked HEK293T cell lysate, holding great potential for genetic analysis and disease prognosis. & Elsevier B.V. All rights reserved.

Keywords: Point mutation K-ras gene Exponential strand displacement amplification Surface enhanced Raman spectroscopy

1. Introduction Point mutation represents a kind of general alterations in the human genome, and it may influence the function of encoded proteins through the transcription and translation processes (Bertina et al., 1994; Halushka et al., 1999; Syvanen, 2001). Recent researches demonstrate that many human diseases including cancer are associated with the point mutation in the particular genes such as single-base substitutions, deletions and insertions (Pharoah et al., 2004; Whibley et al., 2009). The K-ras gene mutations have been observed in many human tumors including lung cancer (Guo et al., 2013), gastric carcinomas (Wu et al., 2004), pancreatic adenocarcinoma (Ebert et al., 2001) and colorectal cancer (Khanna et al., 1999). Moreover, different types of point mutation can affect how human responds to pathogens, chemicals, vaccines and other agents (Evans and Relling, 1999). Therefore, the n

Corresponding author. Fax: þ 86 755 86392299. . E-mail address: [email protected] (C.-y. Zhang). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.bios.2014.10.035 0956-5663/& Elsevier B.V. All rights reserved.

point mutation assay plays an increasingly important role in the medical diagnosis and the clinical therapy. So far, a variety of methods have been developed for the point mutation assay, such as ligase-mediated detection (Landegren et al., 1988; Huang et al., 2009), primer extension-based method (Sokolov, 1990; Litos et al., 2007), flap endonuclease-based cleavage method (Lyamichev et al., 1999; Chen et al., 2005) and polymerase chain reaction (PCR)-based method (Hacia et al., 1998; Fujii et al., 2000; Germer et al., 2000). Among these approaches, PCR-based method dominates the field of point mutation analysis owing to its high amplification efficiency. However, PCR might introduce the false positivity from the nonspecific amplification due to the involvement of complicated thermal cycling steps (Su et al., 2010). Recently, various isothermal amplification reactions have gained increasing attention, among which the strand displacement amplification (SDA) is a representative isothermal technology because of its good specificity and simplicity (Van Ness et al., 2003; Tan et al., 2005; Wang et al., 2011). The SDA method can generate abundant target sequence of interest with the assistance of polymerase and endonuclease, and has been widely

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applied for sensitive detection of nucleic acids, proteins, and small molecules (Zhu et al., 2013; Zhang and Zhang, 2012; Li et al., 2008; Ye et al., 2014). Herein, we develop a new method for sensitive detection of point mutation using exponential SDA-based surface enhanced Raman spectroscopy (SERS). SERS is an emerging and powerful optical technology that can provide a nondestructive and ultrasensitive detection even down to single molecule level (Kneipp et al., 1997; Nie and Emory, 1997; Li et al., 2010). SERS has distinct characteristics of significant signal enhancement, narrow band widths and alleviated photobleaching (Ni et al., 1999; Grubisha et al., 2003; Zhu et al., 2011). Although some SERS-based methods have been reported for point mutation assay (Mahajan et al., 2008; Huh et al., 2009), they usually involve complex chemical modification and thermal cycling with poor sensitivity. In our proposed method, the combination of exponential SDA reaction with SERS provides a simple platform for sensitive detection of point mutation in an isothermal condition. This method can detect human K-ras gene point mutation with a detection limit of 1.4 pM and a large dynamic range of 4 orders of magnitude, and it can even distinguish as low as 1% variant frequency from the mixture of mutant target and wild-type one. Moreover, the proposed method can be applied to analyze the mutant target in the spiked HEK293T cell lysate.

2. Experimental section 2.1. Materials and reagents The oligonucleotides (Table 1) were synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China). The Escherichia coli DNA ligase, the Klenow fragment polymerase, the nicking enzyme of Nt.BbVCI and the deoxynucleotide triphosphates (dNTPs) were purchased from New England Biolabs (Beverly, MA, USA). SYBR Gold was obtained from Xiamen Bio-Vision Biotechnology (Xiamen, China). All other reagents were of analytical grade. 2.2. Preparation of capture probe-modified AuNPs Gold nanoparticles (AuNPs) were prepared by citrate reduction of chloroauric acid following the reported protocol (Brown et al., 2000; Hu and Zhang, 2010). The maximum absorbance wavelength of AuNPs locates at 526 nm, and the concentration of AuNPs is estimated to be about 1.4 nM by assuming the reduction of all

Table 1 Sequences of the targets and the probes.a Note

Sequence (5′–3′)

AAG GCA CTC TTG CCT ACG CCA TCA GCT CCA ACT ACC ACA AGT TT Wild-type target AAG GCA CTC TTG CCT ACG CCA CCA GCT CCA ACT ACC ACA AGT TT Discriminating probe CTT GAC TAG CTA CGA GCT GAG GCT TGA CTA GCT ACG AGC TGA AGG TAG TTG GAG CTG A Hairpin probe PO4-TGG CGT AGG CAA GAG ACA TCG GCC TTT TTT TTT TGG CCG ATG Capture probe SH-(T)9CTT GAC TAG C Reporter probe TAC GAG CTG A-Rox Synthesized trigger TCA GCT CGT AGC TAG TCA AG

Mutant target

a The underlined letters in the mutant target and the wild-type target indicate the point mutation. The italic region in the discriminating probe indicates the recognition sequence of Nt.BbvCI. The Rox in the reporter probe indicates the carboxy-X-rhodamine.

Au3 þ to Au0 and the particle size of 2574 nm (see Fig. S1). The capture probe-modified AuNPs were prepared according to a published protocol with minor modifications (Hurst et al., 2006). Firstly, 1 OD (6.1 nmol) capture probe was added to 1 mL of AuNP solution (9.1 nM), and incubated at room temperature for 16 h. Then the concentration of phosphate (NaH2PO4/Na2HPO4) was adjusted to 10 mM by adding 0.2 M phosphate buffer (pH 7.0), and the concentration of NaCl was adjusted to 0.1 M, followed by standing for 40 h. Lastly, the AuNPs were centrifuged three times to remove the excess capture probes, then resuspended in 234 μL of PBS buffer (10 mM phosphate, 0.1 M NaCl, pH 7.0) and stored at 4 °C before use. In this capture probe-modified AuNPs solution, the concentration of AuNPs was estimated to be 38.9 nM, and the DNA concentration was estimated to be 19.1 μM (see Supplementary information). 2.3. Ligation and SDA reaction The ligation reaction was performed in 10 μL of reaction mixture containing 1  ligase buffer (30 mM Tris–HCl, 4 mM MgCl2, 1 mM DTT, 26 μM NAD þ , 50 μg/mL BSA, pH 8.0), 10 nM discriminating probe, 10 nM hairpin probe, 0.2 U/μL ligase and different concentrations of targets, and incubated at 16 °C for 1 h. After the ligation reaction, the SDA reaction was performed in 10 μL of reaction mixture containing 1 μL of ligation products, 0.75 μL of discriminating probe (1 μM), 0.4 μL of Nt.BbvCI (10 U/ μL), 0.1 μL of polymerase (5 U/μL), 0.25 μL of dNTPs (10 mM), 1 μL of 10  NEB buffer 2 (100 mM Tris–HCl, 500 mM NaCl, 100 mM MgCl2, 10 mM dithiothreitol, pH 7.9), and 6.5 μL of H2O at 37 °C for 30 min, followed by incubation at 80 °C for 20 min to inactivate the enzymes. The amplification products were kept at 4 °C for subsequent analysis. 2.4. Sandwich hybridization reaction and Raman measurement The sandwich hybridization reaction was carried out in the solution containing 10 μL of amplification products, 5 μL of capture probe-modified AuNPs and 5 μL of Rox-labeled reporter probe (10 μM) at the room temperature for 2 h. The solution was then centrifuged at 8000 rpm for 20 min to remove the excess reporter probes. After the removal of the supernatant, the red precipitate was washed three times with 50 μL of 10 mM phosphate buffer (pH 7.0, 0.1 M NaCl) and resuspended in 2 μL of PBS buffer. The droplet was dripped on the silico pellet and air-dried at room temperature before the Raman measurement. The SERS spectra were measured by a LabRAM HR Raman spectrometer with a 632.8-nm laser (HORIBA Jobin-Yvon, France). The laser power at the sample location was 15.7 mW, and the resolution of SERS spectra over 900–1800 cm  1 was about 0.65 cm  1. The collection time for each spectrum was 10 s. 2.5. Gel electrophoresis The amplification products was analyzed using a 10% non-denaturating polyacrylamide gel electrophoresis (PAGE) in 1  TBE buffer (9 mM Tris–HCl, pH 7.9, 9 mM boric acid, 0.2 mM EDTA) at a 100 V constant voltage for 50 min with SYBR Gold as the fluorescent indicator. The stained gel was scanned by a Kodak Image Station 4000 MM (Rochester, NY, USA). The band intensities in the gels were analyzed by Quantity One software. 2.6. Preparation of cell lysate The HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). The cells were removed from the substrate by trypsinization, washed

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twice with phosphate buffered saline (pH 7.4), and pelleted at 2000 rpm for 10 min at 4 °C. Then the cells were resuspended in Western and IP cell lysis solution at a concentration of 5.0  106 cells/mL, and incubated at 4 °C for 30 min. After centrifugation at 12,000 rpm for 30 min at 4 °C, the supernatant was transferred into a fresh tube, flash-frozen and stored at  20 °C before use.

3. Results and discussions 3.1. Principle of point mutation assay As illustrated in Fig. 1, the point mutation assay involves three principal steps: (1) the ligation reaction, (2) the exponential SDA reaction, and (3) the sandwich hybridization. A discriminating probe and a hairpin probe are designed to be partly complementary with the target, and the 3′-terminal nucleotide of the discriminating probe is used to distinguish the mutant target from the wild-type target. In the first step, the mutant target can hybridize with the discriminating probe and the hairpin probe, making 3′-terminal of the discriminating probe and 5′-terminal of the hairpin probe ligate by the ligase. However, the wild-type target cannot lead to the ligation reaction due to the presence of single-base mismatch. In the second step, the 3′-terminal of hairpin probe in the ligated products can act as a primer to initiate the SDA reaction with the assistance of polymerase and nicking enzyme, generating large numbers of triggers. The released trigger can bind to the new discriminating probe to initiate new SDA reactions, leading to an exponential amplification. In the third step, the resultant triggers can hybridize with the capture probe-modified AuNPs and the ROX-labeled reporter probes to form the sandwich hybrids, generating a strong SERS signal. Notably, the formation of sandwich hybrids on the surface of Au NPs can be confirmed by UV–vis absorption and TEM image (see Figs. S2 and S3).

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To demonstrate the feasibility of the proposed method for point mutation assay, we used the non-denaturating PAGE to analyze the SDA products in the presence of mutant target, wild-type target, and the control group without any target, respectively. As shown in Fig. 2A, a well-defined band of triggers (20 nt) is observed in the presence of mutant target (Fig. 2A, lane 1), but no such band is observed in the presence of either wild-type target (Fig. 2A, lane 2) or the control group (Fig. 2A, lane 3), indicating that only mutant target can initiate the SDA reaction. These results are further confirmed by the Raman measurement (Fig. 2B). A strong SERS signal with the characteristic peak of 1504 cm  1 is observed in the presence of mutant target (Fig. 2B, blue line). However, no significant SERS signal is observed in the presence of either wild-type target (Fig. 2B, red line) or the control group without any target (Fig. 2B, black line). The SERS intensity at the 1504 cm  1 characteristic peak can be used for quantitative analysis (Fig. 2C). 3.2. Optimization of ligation reaction The concentration of ligase is a key factor which influences the specificity of ligation reaction and the performance of subsequent SDA reaction. The high-concentration ligase can lead to high ligation efficiency, but it might increase the probability of nick sealing of single-base mismatch, resulting in a nonspecific ligation (Cheng et al., 2012). The low-concentration ligase can ensure a good specificity, but it adversely induces the low ligation efficiency and consequently the poor performance of SDA reaction. To obtain the optimal ligase concentration, we measured the amplified triggers in the presence of mutant target and wild-type target, respectively, using the non-denaturating PAGE (Fig. 3A). When the concentration of ligase increases from 0.05 U/μL to 0.4 U/μL, the band intensity of the amplified triggers in the presence of mutant target (Fig. 3A, lanes 2, 4, 6, 8 and 10) increases correspondingly, but the band of the amplified triggers in the presence of wild-type target (Fig. 3A, lanes 1, 3, 5, 7 and 9) is observed at 0.4 U/μL ligase

Fig. 1. Schematic illustration of point mutation assay using exponential SDA-based SERS.

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Fig. 2. (A) 10% Non-denaturating PAGE analysis of the amplification products of SDA reaction. Lane M is the DNA ladder marker; lanes 1, 2, 3, and 4 represent the amplification products in the presence of 10 nM mutant target, 10 nM wild-type target, the control group without any target, and the synthesized triggers (20 nt), respectively. (B) SERS spectra obtained in the presence of 10 nM mutant target (MT, blue line), 10 nM wild-type target (WT, red line), and the control group without any target (black line). (C) SERS intensity at the 1504 cm  1 characteristic peak in response to 10 nM mutant target (MT, blue color), 10 nM wild-type target (WT, yellow color), and the control group without any target (purple color). Error bars show the standard deviation of three experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. (A) Variance of the amplified triggers with the ligase concentration in the presence of mutant target (lanes 2, 4, 6, 8 and 10) and wild-type target (lanes 1, 3, 5, 7 and 9). The concentration of ligase is 0.05 U/μL (lanes 1 and 2), 0.1 U/μL (lanes 3 and 4), 0.2 U/μL (lanes 5 and 6), 0.3 U/μL (lanes 7 and 8), and 0.4 U/μL (lanes 9 and 10), respectively. Lane M is the DNA ladder marker, and lane 11 is the synthesized triggers (20 nt). (B) Variance of the I/IWT value with the ligase concentration. I and IWT are the band intensity of the amplified triggers in the presence of mutant target (MT) and wild-type target (WT), respectively. (C) Variance of the amplified triggers with the ligation reaction time in the presence of mutant target (lanes 2, 4, 6, 8 and 10) and wild-type target (lanes 1, 3, 5, 7 and 9). The reaction time of ligation is 0.5 h (lanes 1 and 2), 1 h (lanes 3 and 4), 1.5 h (lanes 5 and 6), 2 h (lanes 7 and 8), and 2.5 h (lanes 9 and 10), respectively. Lane M is the DNA ladder marker, and lane 11 is the synthesized triggers (20 nt). (D) Variance of the I/IWT value with the ligation reaction time. I and IWT are the band intensity of the amplified triggers in the presence of mutant target (MT) and wild-type target (WT), respectively. The concentration of mutant target is 10 nM, and the concentration of wild-type target is 10 nM. Error bars show the standard deviation of three experiments.

as well (Fig. 3A, lane 9). For quantitative analysis, we investigated the variance of I/IWT value with the ligase concentration (Fig. 3B), where I and IWT are the band intensity of the amplified triggers in the presence of mutant target and wild-type target, respectively.

The value of I/IWT increases with the increasing concentration of ligase from 0.05 U/μL to 0.2 U/μL, followed by the decrease beyond the concentration of 0.3 U/μL. Thus, 0.2 U/μL ligase is used in the subsequent research.

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Fig. 4. (A) Variance of the amplified triggers with the template concentration in the presence of mutant target (lanes 2, 4, 6, 8 and 10) and in the control group (lanes 1, 3, 5, 7 and 9). The concentration of template is 25 nM (lanes 1 and 2), 50 nM (lanes 3 and 4), 75 nM (lanes 5 and 6), 100 nM (lanes 7 and 8), and 150 nM (lanes 9 and 10), respectively. Lane M is the DNA ladder marker, and lane 11 is the synthesized triggers (20 nt). (B) Variance of the I/I0 value with the template concentration. I and I0 are the band intensity of the amplified triggers in the presence of mutant target (MT) and in the control group, respectively. (C) Variance of the amplified triggers with the nicking enzyme concentration in the presence of mutant target (lanes 2, 4, 6, 8 and 10) and in the control group (lanes 1, 3, 5, 7 and 9). The concentration of nicking enzyme is 0.2 U/ μL (lanes 1 and 2), 0.3 U/μL (lanes 3 and 4), 0.4 U/μL (lanes 5 and 6), 0.5 U/μL (lanes 7 and 8), and 0.6 U/μL (lanes 9 and 10), respectively. Lane M is the DNA ladder marker, and lane 11 is the synthesized triggers (20 nt). (D) Variance of the I/I0 value with the nicking enzyme concentration. I and I0 are the band intensity of the amplified triggers in the presence of mutant target (MT) and in the control group, respectively. The concentration of mutant target is 10 nM. Error bars show the standard deviation of three experiments.

In addition, the reaction time of ligation influences the specificity as well (Cheng et al., 2012). As shown in Fig. 3C, the band intensity of the amplified triggers in the presence of mutant target (Fig. 3C, lanes 2, 4, 6, 8 and 10) reaches the maximum at the reaction time of 1 h (Fig. 3C, lane 4), and the band of the amplified triggers in the presence of wild-type target (Fig. 3C, lanes 1, 3, 5, 7 and 9) is observed at the reaction time of 2 h as well (Fig. 3C, lane 7). For quantitative analysis, we investigated the variance of I/ IWT value with the ligation time (Fig. 3D), where I and IWT are the band intensity of the amplified triggers in the presence of mutant target and wild-type target, respectively. The value of I/IWT increases with the increase of ligation time from 0.5 h to 1 h, followed by the decrease beyond the ligation time of 1 h. Therefore, the reaction time of 1 h is selected for ligation in the subsequent research. 3.3. Optimization of SDA reaction In SDA reaction, the template concentration is crucial to the amplification efficiency (Zhang and Zhang, 2012). In order to obtain the high amplification efficiency, the concentration of template (i.e. discriminating probe in this experiment) was experimentally optimized. As shown in Fig. 4A, the amplified triggers in the control group (Fig. 4A, lanes 1, 3, 5, 7 and 9) cannot be observed at different template concentrations. However, the

amplified trigger in the presence of mutant target (Fig. 4A, lanes 2, 4, 6, 8 and 10) increases with the increasing template concentration, followed by the decrease beyond the concentration of 75 nM (Fig. 4A, lane 6). For quantitative analysis, we investigated the variance of I/I0 value with the template concentration (Fig. 4B), where I and I0 are the band intensity of the amplified triggers in the presence of mutant target and in the control group, respectively. The value of I/I0 increases with the increasing template concentration from 25 nM to 75 nM, followed by the decrease beyond the concentration of 75 nM. Thus, 75 nM template is used in the subsequent research. The amount of nicking enzyme is also of great importance to the amplification efficiency of SDA reaction (Liang et al., 2004). As shown in Fig. 4C, the amplified triggers in the control group (Fig. 4C, lanes 1, 3, 5, 7 and 9) are nearly unobservable at different concentrations of nicking enzyme. In contrast, the amplified trigger in the presence of mutant target (Fig. 4C, lanes 2, 4, 6, 8 and 10) increases with the increasing template concentration, followed by the decrease beyond the concentration of 75 nM (Fig. 4C, lane 6). For quantitative analysis, we investigated the variance of I/I0 value with the concentration of nicking enzyme (Fig. 4D), where I and I0 are the band intensity of the amplified triggers in the presence of mutant target and in the control group, respectively. The value of I/ I0 increases with the increasing concentration of nicking enzyme from 0.2 U/μL to 0.4 U/μL, followed by the decreases beyond the

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Fig. 5. (A) SERS spectra in the presence of mutant target in the range from 0 to 10 nM. (B) Linear relationship between the SERS intensity at 1504 cm  1 peak and the logarithm of mutant target concentration. Inset shows the SERS spectra from 20 randomly selected spots in one droplet sample with and without 10 nM mutant target, respectively. Error bars show the standard deviation of three experiments.

nicking enzyme concentration of 0.4 U/μL. Therefore, 0.4 U/μL nicking enzyme is used in the subsequent research. 3.4. Sensitivity of the proposed method To investigate the capability of the proposed method for point mutation assay, we measured the mutant target with different concentrations under the optimized experimental conditions. As shown in Fig. 5A, the SERS signal improves with the increase of mutant target from 0 to 10 nM. The Raman intensity at 1504 cm  1 peak is used for quantitative analysis. In the logarithmic scale, the Raman intensity has a linear correlation with the concentration of mutant target over a range from 3.2 pM to 10 nM (Fig. 5B). The correlation equation is I ¼11.146þ1307.166 log10 C (R ¼0.987), where I is the Raman intensity at 1504 cm  1 peak and C is the concentration of mutant target (pM). The detection limit of 1.4 pM is obtained by evaluating the average response of the control group plus three times standard deviation. The sensitivity of the proposed method is superior to previous SERS-based method (20 pM) (Huh et al., 2009), the cationic conjugated polyelectrolytebased fluorescence method (30 pM) (Wang et al., 2012), AuNPsbased colorimetric assay (20 pM) (Valentini et al., 2013), and the

Fig. 6. (A) The SERS intensity at 1504 cm  1 peak as a function of variant frequency. The total concentration of mutant target and wild-type target is 5 nM. (B) The correlation of measured and added mutant targets in the cell lysate. The concentration of added mutant target is 0.1 nM, 1.0 nM, and 10.0 nM, respectively. Error bar show the standard deviation of three experiments.

toehold-mediated strand displacement-based electrochemistry method (58 pM) (Gao et al., 2014). Notably, this method demonstrates a good reproducibility with almost overlapping spectra for 20 randomly selected spots and the relative standard deviation (RSD) of 4.3% for the detection of 10 nM mutant target (inset of Fig. 5B). 3.5. Measurement of variant frequency and detection of the spiked cell sample At the early stage of tumor genesis, there is only a minimal amount of mutant cells which are often masked by the presence of large amounts of wild-type cells (Hashimoto et al., 2005). Therefore, it is important to estimate the variant frequency of point mutation in genes. To evaluate the capability of the proposed method for point mutation assay, we measured the SERS intensity of 5 nM mixture containing 0%, 1%, 5%, 10%, 50% and 100% mutant target, respectively. Fig. 6A shows the SERS intensity improves with the increase of variant frequency in the mixture. Notably, this method can distinguish as low as 1% variant frequency. To demonstrate the capability of the proposed method for real sample analysis, we further measured the spiked mutant target in 10% HEK293T cell lysate. As shown in Fig. 6B, different

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Table 2 Analytical recovery of mutant target added to the cell lysate sample (n¼ 3). Note

Added (nM)

Measured (nM)

Recovery (%)

RSD (%)

Mutant target

0 0.1 1.0 10.0

0 0.09 0.917 9.40

– 90.0 91.7 94.0

– 3.10 4.60 5.03

concentration of mutant target in the cell lysate can be accurately determined, and a good linear relationship is obtained between the measured concentration and the added concentration. With the addition of 0.1 nM, 1 nM and 10 nM mutant target, the measured recovery is in the range from 90.0% to 94.0% with the RSD from 3.10% to 5.03% (Table 2), indicating that the proposed method can be applied for real sample analysis with good reliability.

4. Conclusions In summary, we have developed a new method for sensitive detection of point mutation using exponential SDA-based SERS. Due to the excellent specificity of discriminating probe and E. coli DNA ligase (Wang et al., 2010), the proposed method can distinguish the point mutation between the mutant target and the wildtype target. Taking advantage of the high amplification efficiency of exponential SDA and the intrinsically high signal enhancement of SERS, this method can sensitively measure the mutant target with a detection limit of 1.4 pM and a large dynamic range of 4 orders of magnitude from 3.2 pM to 10 nM, and it can even discriminate as low as 1% variation frequency from the mixture of mutant target and wild-type target. Importantly, the proposed method can be applied for real sample analysis, and might be further extended to detect multiplex point mutation locus by simply designing new discriminating probes and using different Raman dyes.

Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant nos. 21325523 and 21205128), the Award for the Hundred Talent Program of the Chinese Academy of Sciences, the Guangdong Innovation Research Team Fund for Lowcost Healthcare Technologies, and the Fund for Shenzhen Engineering Laboratory of Single-molecule Detection and Instrument Development (Grant no. (2012) 433).

Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at.

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Sensitive detection of point mutation using exponential strand displacement amplification-based surface enhanced Raman spectroscopy.

Accurate identification of point mutation is particularly imperative in the field of biomedical research and clinical diagnosis. Here, we develop a se...
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