Dual-Amplification Strategy-Based SERS Chip for Sensitive and

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Dual-Amplification Strategy-Based SERS Chip for Sensitive and Reproducible Detection of DNA Methyltransferase Activity in Human Serum Runzhi Chen, Huayi Shi, Xinyu Meng, Yuanyuan Su, Houyu Wang, and Yao He Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05595 • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 6, 2019

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Analytical Chemistry

Dual-Amplification Strategy-Based SERS Chip for Sensitive and Reproducible Detection of DNA Methyltransferase Activity in Human Serum Runzhi Chen,‡ Huayi Shi,‡ Xinyu Meng, Yuanyuan Su, Houyu Wang* and Yao He* Laboratory of Nanoscale Biochemical Analysis, Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano & Soft Materials (FUNSOM), and Collaborative Innovation Center of Suzhou Nano Science and Technology (NANO-CIC), Soochow University, Suzhou, Jiangsu 215123, China *E-mail: [email protected]; [email protected]; Fax: 86-512-65880946.

ABSTRACT: Herein we present a dual-amplification sensing strategy-based surface-enhanced Raman scattering (SERS) chip, which combines rolling circle amplification (RCA) and Poly adenine (Poly A) assembly for sensitive and reproducible determination of the activity of M.SssI, a cytosine-guanine dinucleotides (CpG) methyltransferase (MTase). Typically, in the presence of M.SssI, RCA process is triggered, resulting in long, single-stranded DNA (ssDNA) fragments that are hybridized with thousands of Raman reporters of Cy3. Afterwards, the resultant ssDNA fragments are conjugated to SERS-active substrates made of silver core-gold satellite nanocomposites-modified silicon wafer (Ag-Au NPs@Si), with the SERS enhancement factor of ∼5 × 106. The core-satellite nanostructures are assembled relied on the strong affinity of Poly A towards gold/silver surface. Of particular significance, the developed SERS chip displays an ultrahigh sensitivity with a low limit of detection (LOD) of 2.8 × 10-3 U/mL, which is around two orders of magnitude higher than most reported methods. In addition, the constructed chip features a broad detection range covering from 0.05 to 50 U/mL. Besides for the ultrahigh sensitivity and broad dynamic range, the chip also features good reproducibility (e.g., the relative standard deviation (RSD) is less than ~12%). Taking advantages of these merits, the developed chip is feasible for accurate discrimination of M.SssI with various concentrations spiked in human serum samples with good recoveries ranging from 99.6% to 107%.

efforts have been devoted to develop new detection methods (e.g. fluorometry10-13, surface-enhance Raman scattering (SERS) assays14, colorimetry15-18, electrochemical assays19,20) for sensitive and specific discrimination of MTase activity. The most popular sensing strategies employed by these methods are DNA-based signal amplification strategies, including polymerase chain reaction21, nicking enzyme signal amplification (NESA)22, strand displacement amplification (SDA)12,23, and rolling circle amplification (RCA)12,24,25. Among them, RCA is an isothermal amplification technique in which DNA primer can be continuously amplified along the circular template by the polymerase, achieving a 105-fold linear and a 109fold or even more exponential amplification26,27. Based on these, RCA has gained an ultrahigh sensitivity in the detection of DNA MTase12,25. Despite these progresses, it remains a major challenge for discriminating DNA MTase in real samples with both high sensitivity and reliable reproducibility.

INTRODUCTION DNA methylation, a most commonly epigenetic behavior, is closely linked with the regulation of gene expression1. Normally, methylated cytosine-guanine dinucleotides (CpG) reaches around 60-80% in human genome2. The process of DNA methylation involves transferring of methyl group from S-adenosyl-Lmethionine (SAM) to C-5 position of cytosine in CpG islands. Such process is specifically catalyzed by DNA methyltransferase (MTase)3,4. Particularly, in the process of cancerous transformations, the tumor suppressor genes (e.g., p53) are hypermethylated while many oncogenes are hypomethylated5,6. Up to present, aberrant DNA MTase activity has been commonly detected in cancer initiation and progression7,8. Therefore, there is a great demand for the development of a sensitive, selective and reproducible method for determining DNA MTase activity. The traditional standard approach of detecting DNA MTase activity is a radioactive assay based on [methyl3H]-labelled SAM9. To avoid radioactive hazards, many

Another prominent characteristic of RCA technique is its benign compatibility with on-chip analysis because its

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Figure 1. Schematic illustration of dual-amplification strategy of rolling circle amplification (RCA) combined with Poly adenine (Poly A) assembly for sensitive and reproducible detection of DNA methyltransferase activity. As a proof of concept, Escherichia coli (E.coli) CpG methyltransferase M.SssI (M.SssI) is selected as the target. Herein, we develop a novel dual-amplification sensing strategy for sensitively and reproducibly detecting Escherichia coli (E.coli) CpG MTase M.SssI (M.SssI) activity. The developed strategy combines RCA with Poly A assembly. In RCA process, upon the addition of M.SssI, the long single-strand DNA (ssDNA) sequences hybridized with numerous Raman reporters can be achieved under isothermal conditions. In the Poly A assembly, core (Ag)-satellite (Au) nanoparticles (Ag-Au NPs) modified on silicon wafer (Ag-Au NPs@Si) is fabricated relied on the strong affinity of Poly A towards gold/silver surface, also producing abundant SERS hot spots. As such, the RCA product of ssDNA is linked to the surface of nanohybrids to construct the SERS chip, generating prominent SERS signals. As a result, the limit of detection (LOD) in the constructed SERS chip is as low as 2.8 × 10-3 U/mL of M.SssI. In addition, the developed SERS chip has adaptive reproducibility (RSD ~12%), good selectivity against other kind of DNA MTase (e.g., Dam), and broad dynamic range (e.g., 0.05~50 U/mL). More importantly, the developed chip can be used to detect various concentrations of M.SssI incorporated into real samples (e.g., human serum), indicating potential feasibility of the developed chip for clinical applications.

product can be immobilized on a solid support (e.g., glass, microplate, etc.)27-29. Such property inspires us to combine RCA with another chip-based signal amplification strategy to realize more sensitive assay. Surface-enhanced Raman scattering (SERS) chip is a rising candidate, which is made of plasmonic nanostructures modified on a planar solid support30-32. More recently, Poly adenine (Poly A)-mediated selfassembly strategy has gained much attention, which can fabricate metallic nanocomposites with a controllable plasmonic property33-41. Distinguished from only one contacting site of thiolated DNA towards gold/silver surface, Poly A block has numerous binding sites, which is composed of consecutive adenines34-41. As a result, the distribution density of Poly A-linked plasmonic nanostructures on gold/silver surface can be tuned by adjusting the number of adenine, thus regulating the localized surface plasmon resonance (LSPR) of nanocomposites. In optimal conditions, the SERS enhancement factor (EF) of Poly A-assembled SERS chip can be up to ∼107. On the other aspect, Poly A-assembled SERS chip features adaptable reproducibility in the analysis of targets in real systems (e.g., the value of relative standard deviation (RSD) is less than 15%) due to its robust plasmonic nanostructures tightly immobilized on solid support, effectively preventing random motion and uncontrolled agglomeration of plasmonic nanostructures in liquid phase34,37. Bearing this in mind, it is possible to integrate RCA strategy with Poly A assembly strategy to construct a sensitive, reproducible platform for detecting DNA MTase activity in real samples.

EXPERIMENTAL SECTION Preparation of SERS substrates. First, satellite Au NPs (~13 nm) were prepared using the standard citrate reduction method. Second, silver nanoparticles decorated silicon support (Ag NPs@Si) was prepared via silicon-


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Analytical Chemistry progressive recognition sites is designed: the first recognition site for DNA MTase (e.g., M.SssI), the second recognition site for methylation sensitive restriction endonuclease (e.g., HpaII) and the last recognition site for circular DNA template (e.g., padlock DNA probe) to trigger RCA reactions. The detailed dsDNA sequence is listed in Supporting Information (see Primer 1 and Primer 2 in Table S1). In principle, when M.SssI exists in the detected samples (e.g., human serum samples), the unmethylated dsDNA (5’-CCGG-3’) is catalyzed as hypermethylated dsDNA (5’-CCmGG-3’), which can not be cleaved by HpaII. Furthermore, the single stranded tail of dsDNA binds to the padlock DNA to further trigger the RCA process. In this process, a long single-strand DNA (ssDNA) composed of tandem repetitive units is produced upon addition of DNA polymerase of Phi29 and dNTPs. In the following steps, multiple Raman reporters (e.g., Cy3) labeled complementary segments are hybridized with asproduced ssDNA. The other end of dsDNA is tagged with thiol group, which can be immobilized on the surface of silver nanoparticles modified silicon wafer (Ag NPs@Si), producing strong SERS signals. Ag NPs@Si is readily prepared based on the reported silicon-assisted galvanic displacement method 31,32. While in the absence of M.SssI, the dsDNA (5’-CCGG-3’) would not be methylated, thus it can be cleaved into two fragments by methylation sensitive restriction endonuclease of HpaII.

assisted galvanic displacement method31,32, in which the silicon wafer was soaked in the 1.25 mM AgNO3 solution, followed by the treatment with 5% HF (v/v) for 30 minutes. Afterwards, DNA sequences of Poly A30 (30 repeatable adenines at the 5’ end)-P1 and Poly A30-P2 (the complementary DNA of P1) were respectively conjugated to the as-resultant Ag NPs@Si and Au NPs in phosphate buffer (pH 7.0) for 16h. The detailed sequences of P1 and P2 were listed in Table S2. Ultimately, Ag core-Au satellite nanostructures immobilized on silicon wafer (Ag-Au NPs@Si) was obtained when Poly A30-P1 linked Ag NPs@Si were mixed with Poly A30-P2 linked Au NPs in the hybridization buffer (0.3 M PBS, pH 7.0) for 24 h. Methylation and Cleavage of dsDNA Probe. The dsDNA probe was methylated in 15 μL of methylation buffer which contains M.SssI with various amounts, 2 μM dsDNA probe, 1 × NEB buffer (50 mM NaCl, 10 mM TrisHCl (pH 7.9), 10 mM MgCl2, 1 mM DTT), and 160 μM SAM. The reaction solution was incubated at 37 oC for around 2 h, followed by inactivation at 65 °C for nearly 20 min. For cleavage reaction, 20 μL of cleavage buffer containing 1 × Cut Smart buffer (50 mM KAc, 20 mM Tris, 10 mM Mg(Ac)2, 100μg BSA) and 500 U/mL E.coli restriction endonuclease HpaII (HpaII) was mixed with 15 μL of methylated dsDNA at 37 °C for 1 h, followed by inactivation at 80 °C for around 20 min.

In order to achieve more SERS enhancements, Poly Aassisted assembly is employed for constructing more complex SERS substrates in a controllable manner, showing more distinct SERS effects. As shown in blue dashed frame in Figure 1, two single-strand DNA sequences containing 30 consecutive adenines (Poly A30, pink regions) at 5’ end are respectively modified on the surface of Ag NPs@Si and the surface of gold nanoparticles (Au NPs). Then, the asymmetric nanostructure of Ag-Au NPs@Si can be constructed by the hybridization reaction among these two DNA strands. Distinguished from only one contacting site of thiolated DNA towards gold/silver surface, Poly A block has numerous binding sites. Thereby, the SERS effect of nanocomposites could be tuned by adjusting the length of Poly A (e.g., Poly A10, Poly A30, Poly A50, etc.). As previously reported, the SERS enhancement factor (EF) values of SERS substrates prepared by Poly A10, Poly A30 and Poly A50 are 12.8 × 106, 5.4 × 106 and 3.1 × 106, respectively34. In this study, Poly A30 is selected as the optimal length of Poly A block taking into account its two advantages: assembly of more Au NPs (e.g., ~15-20 Au NPs per Ag NP) and relatively high assembly efficiency (~90%)34,37. Compared with Ag NPs@Si, Ag-Au NPs@Si has more SERS hotspots ascribed from more plasmonic nanoparticles linked to core Ag NP, thereby improving SERS enhancement and realizing ultrahigh sensitivity. On the other aspect, Ag-Au NPs@Si has a larger surface area

Determination of M.SssI activity. The reaction solution for RCA contains 25 μL of Tris-HCl buffer (50 mM, pH 7.5), (NH4)2SO4(10 mM), MgCl2(10 mM), DTT(4 mM), deoxyribonucleotide triphosphates (dNTPs) (200 μM), circular probe(30 nM), 4 U/mL phi29 DNA polymerase, and 1 μL products of methylation and cleavage reaction with different M.SssI concentrations (0, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, and 200 U/mL). The reaction was performed at 30 °C for 30 min. To achieve the conjugation between DNA and SERS substrates, 0.5 μL of RCA products of ssDNA in the assembly buffer (10 mM phosphate, pH 7.0) was dropped onto Ag-Au NPs@Si surface for 12 h. The interfering sample of Dam was also detected under the same conditions. As for the determination of M.SssI activity in human serum samples, the collected human blood samples were centrifuged at a low speed of 3500 rpm/min for 15 min to obtain serum samples. Then the serum samples were spiked with M.SssI with four different concentrations (0, 0.5, 5.0 and 50 U/mL). The Raman spectra were detected by using a Raman microscope (633 nm laser, 100 × objective, 1 sec of acquisition time, 0.2 mW of acquisition time).

RESULTS AND DISCUSSION Dual-amplification strategy. The dual-amplification strategy of RCA combined with Poly A assembly is schematically illustrated in Figure 1. In the RCA system, a double-stranded DNA (dsDNA) probe with three



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reproducibility of SERS signals obtained from the developed chip are primarily investigated, as revealed in Figure 2e and 2f. The Raman intensity of R6G (10-4 M) in Ag-Au NPs@Si is ~6.6 × 103-fold stronger than those in pure silicon substrate, and ~2.3-fold stronger than those in Ag NPs@Si (Figure 2e). Therefore, SERS enhancement factor (EF) of Ag-Au NPs@Si is ∼5.0 × 106, ∼2-time higher than that of Ag NPs@Si (e.g., ∼2.1 × 106) under the identical conditions. The detailed process of EF calculation is given in Supporting Information. Moreover, the as-prepared SERS chip shows a good reproducibility. As shown in Figure 2f, uniform Raman spectra of R6G are observed. The RSD value of Raman intensities of R6G at 1364 cm−1 is as low as ~4.4%.

than Ag NPs@Si, which could provide more binding sites towards RCA products.

Figure 2. Characterizations of SERS chip. (a) The photo of silver nanoparticles modified silicon wafer (Ag NPs@Si, top) and silver core-gold satellite nanohybrids modified on silicon wafer (Ag-Au NPs@Si, down) (0.3 cm × 0.3 cm). (b) The corresponding SEM image of the Ag NPs@Si. Inset represents zoom-in SEM image of one Ag NP. (c) The corresponding SEM image of Ag-Au NPs@Si. Inset represents zoom-in SEM image of one core-satellite nanostructure. (d) UV-vis spectra of Au NPs (black), Ag NPs@Si (blue), and Ag-Au NPs@Si (red). (e) Raman spectra of R6G (10-4 M) distributed on pure silicon wafer (black), Ag NPs@Si (red) and Ag-Au NPs@Si (blue). (f) SERS mapping spectra of 10-4 M R6G collected from 50 random spots on the surface of chip. Excitation wavelength: 633 nm; laser power: 0.2 mW.

Figure 3. (a) Schematic illustration of the role of HpaII in RCA strategy. (b) Agarose gel electrophoresis (AGE) analysis of the digestion products of M.SssI and HpaII. The products are separated by 3% agarose gel electrophoresis and stained by Gel red. Lane 1 is the dsDNA; Lane 2 represents the products in the presence of dsDNA and HpaII; Lane 3 represents the products in the presence of dsDNA, M.SssI, and HpaII. (c) Electrophoretic identification of amplification products of the RCA reaction in the presence of Phi29 DNA polymerase.

Characterizations of SERS chip. The size of asprepared SERS chip is 0.3 cm × 0.3 cm (Figure 2a). As shown in SEM images in Figure 2b and 2c, the large Ag NPs with the size of ~120 nm are unifromly distributed. Moreover, as shown in the zoom-in SEM image in Figure 2c, the surface of Ag NP is linked with several small Au NPs with the size of ~13 nm (Figure S1). Statistically, around 15 Au NPs are distributed around each Ag NP. As displayed in Figure 2d, there are two typical absorption peaks located at 420 nm and 520 nm in the UV spectrum of Ag-Au NPs@Si (red curve), which are respectively assigned to Ag NPs and Au NPs.37 The intensity and

In the RCA design, the role of HpaII is cleaving unmethylated dsDNA (5’-CCGG-3’) rather than hypermethylated dsDNA (5’-CCmGG-3’), as illustrated in Figure 3a. To interrogate the significant role of HpaII in RCA strategy, 3.0% agarose gel electrophoresis is performed by using Gel red as the indicator. As observed in Figure 3b, there is a new band of 38-bp at lane 2, indicating the occurrence of cleavage reaction on unmethylated dsDNA by HpaII when M.SssI is absent. When both M.SssI and HpaII are present, only one band of the original probe exists, suggesting no cleavage


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reaction takes place (Figure 3b, lanes 3). The final amplification products are further analyzed by 3.0% agarose gel electrophoresis (Figure 3c). The distinct band appears in the presence of Phi29 DNA polymerase, but no obvious band can be observed in the control group without Phi29, indicating the presence of Phi29 polymerase would trigger the RCA reaction.

related to the amplification efficiency of RCA. Afterwards, we investigated the composition of RCA reaction buffer. As shown in Figure S3b, the RCA products are not observed in lane 1 (1 × NEBuffer) and lane 2 (1 × CutSmart Buffer), while the RCA products appear in lane 3 (1 ×phi29 DNA Polymerase Reaction Buffer) and lane 4 (a mixture of NEBuffer, CutSmart Buffer and phi29 DNA Polymerase Reaction Buffer). Thus, RCA reaction can proceed normally in the mixed buffer solution. Furthermore, we further investigated the effect of RCA reaction time on the assay. As displayed in Figure S3c, the Raman intensity reaches the maximum value when the RCA reaction time is 30 min. In this case, the dNTPs in the solution have been reacted, so the reaction cannot proceed. Ultimately, Cy3 labeled detection probes were used as output signals. The hybridization time between the Cy3 labeled detection probes and RCA products was also optimized. As shown in Figure S3d, the Raman signal intensity increases obviously with the increase of hybridization time from 0.5 to 12 h. Further increase of the hybridization time, Raman signal intensity levels off. This phenomenon is due to the saturation of Cy3 labeled detection probe binding with RCA products. Based on above results, the hybridization time of 12 h is employed in the following experiments.

Optimization of SERS chip. To achieve the best analytical performance, methylation time, HpaII cleavage time, composition of RCA reaction buffer, RCA reaction time, and hybridization time of Cy3 labelled detection probes were optimized respectively. To investigate the effects of methylation time on final Raman spectra of Cy3, dsDNA was treated with M.SssI for different times ranged from 0 to 4.0 h before the incubation with HpaII and Phi29. As revealed in Figure S2, when the methylation time reaches ~2 h, the maximal SERS intensity achieves, implying methylation reaction arrives at the steady state at ~2 h triggered by M.SssI. Accordingly, the appropriate methylation time is 2.0 h. HpaII cleavage time also plays key roles in subsequent cleavage process. Next, the effects of HpaII cleavage time on SERS spectra were studied, as

Figure 4. SERS spectra of the M.SssI (0 U/mL, 50 U/mL) collected from Ag-Au NPs@Si (red) and Ag NPs@Si (blue). Inset represents corresponding Raman intensities of Cy3 at 1595 cm−1. All error bars show the standard deviation determined from three independent assays. (excitation wavelength = 633 nm, acquisition time = 1 sec, laser power =0.2 mW)

Figure 5. (a) SERS spectra of Cy3 of M.SssI with different concentrations (e.g., 0 U/mL, 0.05 U/mL, 0.1 U/mL, 0.5 U/mL, 1 U/mL, 5 U/mL, 10 U/mL, 50 U/mL, 100 U/mL and 200 U/mL) collected from Ag-Au NPs@Si. (b) Corresponding Raman intensities of Cy3 at 1595 cm-1. Inset represents the histogram of SERS intensity in 0.05 to 50 U/mL. (c) The linear fitting of Raman intensity (at 1595 cm-1) with logarithmic M.SssI concentrations from 0.05 to 50 U/mL. All error bars show the standard deviation determined from three independent assays. (excitation wavelength = 633 nm, acquisition time = 1 sec, laser power = 0.2 mW)

revealed in Figure S3a. The Raman intensity gradually decreases along with increasing HpaII cleavage time from 0 to 1.0 h. The Raman intensity no longer changes significantly after 1.0 h reaction because almost all dsDNA in the system have been cleaved at such time. As thus, the optimized HpaII cleavage time is 1.0 h. The composition of RCA reaction buffer and RCA reaction time are closely



Detection of M.SssI in standard samples. Under the optimized experimental conditions, the dynamic range and detection sensitivity of the proposed SERS chip for M.SssI assay were evaluated. As displayed in Figure 4, upon addition of M.SssI (50 U/mL), distinct SERS spectra are measured in SERS chips of Ag-Au NPs@Si and Ag NPs@Si. In contrast, much weak spectra are detected in the control groups (0 U/mL of M.SssI). Quantitatively, the SERS intensity at 1595 cm−1 in Ag-Au NPs@Si is around 2.0-time stronger than that in Ag NPs@Si (see inset in Figure 4). Next, the reproducibility of SERS signals in the detection of 0.5 and 50 U/mL M.SssI is investigated, as exhibited in Figure S4. The identical SERS mapping spectra collected from random 30 spots on chip surface together with the corresponding low RSD values less than ~12% well support adaptable reliability of the developed SERS chip.

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Figure 6. Selectivity of the proposed method. (a) SERS spectra of Cy3 of distributed on control (black), M.SssI (red), and Dam (blue). (b) Corresponding Raman intensities of Cy3 at 1595 cm-1. The concentration of Dam MTase is 50 U/mL, and the concentration of M.SssI MTase is 50 U/mL. All error bars show the standard deviation determined from three independent assays. (excitation wavelength = 633 nm, acquisition time =1 sec, laser power = 0.2 mW)

When the M.SssI solution (0.05-50 U/mL) drops onto the chip, the SERS signals of Cy3 are getting stronger (Figures 5a) and their corresponding intensities at 1595 cm−1 also enhance (Figures 5b) when the concentration of M.SssI grows. Typically, as shown in Figure 5c, a good linearity exists between the logarithmic concentration of M.SssI and the normalized Raman intensity (ICy3/IBG). The corresponding regression equation is y = 1.0565 + 3.7705 log10x (correlation coefficient R2 = 0.996). Remarkably, the limit of detection (LOD) is calculated to be 2.8 × 10-3 U/mL by setting the signal(S)-to-noise(N) ratio of 3:1(S/N=3) (see the detailed calculation process in Supporting Information). Notably, compared with other reported methods for the detection of DNA MTase activity ranging from 0.05 to 10 U/mL16, the presented SERS chip has around two orders of magnitude higher sensitivity (see Table S2). The ultrahigh sensitivity is ascribed to the dual-amplification strategy in the combination of RCA and Poly A, generating a myriad of SERS hot spots. To further confirm the contribution of Poly A to improve analytical performance, the simple nanostructure of Ag NPs@Si combined with RCA for discrimination of M.SssI under the same conditions is selected as a comparison. As revealed in Figure S5, the SERS chip of Ag NPs@Si has a relatively poor sensitivity with the LOD of 0.45 U/mL compared with Ag-Au NPs@Si (e.g., 2.8 × 10-3 U/mL) due to its lower SERS enhancement effect. On the other aspect, compared with other presented approaches, the SERS chip based on the dual-amplification strategy features more binding sites to RCA products, also yielding a wider detection range from 0.05 to 50 U/mL (also see Table S2). As for Ag NPs@Si, its dynamic range is from 2 to 50 U/mL (Figure S5), much narrower than that of Ag-Au NPs@Si, which is resulted from the fact that the surface area of Ag-Au NPs@Si assembled by Poly A is ~1.4 times larger than that of Ag NPs@Si. The detailed process of surface area calculation is introduced in Supporting Information.

To validate the selectivity of the proposed strategy for M.SssI activity assay, the Raman spectra in the presence of another kind of DNA MTase, DNA adenine MTase (Dam) were tested. As indicated in Figure 6, only M.SssI group has obvious Raman intensity of Cy3 at 1595 cm-1, whereas the interfering group has very weak Raman signals, comparable to that of control group. These results suggest that the proposed strategy possesses a good selectivity toward M.SssI against other interfering DNA MTase, which is ascirbed to the specific site recognition of MTase toward their substrate in the developed system. In addition, the as-proposed method also can be used for screening of DNA MTase inhibitor and inhibitory activity, as shown in Figure S6.

Figure 7. SERS spectra of Cy3 of distributed on control (red) and human serum sample (blue) spiked with different concentrations of M.SssI MTase, respectively.


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Discrimination of MTase activity in human serum samples. In order to evaluate the feasibility of the developed method for monitoring DNA MTase in real samples, recovery tests of M.SssI in human serum samples were performed using the standard addition method. Four different concentrations of M.SssI at 0, 0.5, 5.0 and 50 U/mL were separately spiked into 10% diluted human serum samples42. As depicted in Figure 7, it is obvious that the Raman intensity for four different concentrations of M.SssI is comparable in both diluted human serum sample and buffer solution. The recoveries of the four samples including 0 U/mL, 0.5 U/mL, 5.0 U/mL and 50 U/mL M.SssI are 103%, 107%, 102%, and 99.6% with RSD of 2.1%, 3.8%, 4.2% and 2.4%, respectively. These results indicate the potentiality of the method for the accurate quantification of M.SssI in real biological samples.

Author Contributions ‡These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We express our grateful thanks to Prof. Shuit-Tong Lee's general help and valuable suggestion. The authors appreciate financial support from the National Natural Science Foundation of China (21825402, 31400860, 21575096, and 21605109), 111 Project and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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CONCLUSIONS In summary, we have developed a dual amplification SERS sensing strategy combining RCA and Poly A assembly for ultrasensitive detection of DNA methyltransferase. Due to the high amplification efficiency of the developed strategy, the proposed method exhibits a ultrahigh sensitivity toward M.SssI with the detection limit down to 2.8 × 10-3 U/mL, which is around two orders of magnitude higher than those of most reported methods for the detection of DNA MTase activity. In addition, the presented method also features a good reproducibility with the RSD value less than ~12%. Taking advantages of these features, the developed SERS sensing strategy is efficacious for the determination of M.SssI spiked in human serum samples with high recoveries more than 99.6%, suggesting potential promise for practical applications in early cancer diagnosis.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:... Reagents and apparatus, Calculation of SERS enhancement factor (EF), Calculation of limit of detection (LOD), Calculation of surface area, Characterization of Au NPs (Figure S1), Dynamic study of M.SssI activity (Figure S2), Optimization of experimental condition (Figure S3), SERS reproducibility in the detection of M.SssI (Figure S4), Determination of M. SssI activity by Ag NPs@Si (Figure S5), Assay of the inhibition of M.SssI activity (Figure S6), DNA sequences (Table S1), Comparison of recently reported method of M.SssI assay (Table S2).

AUTHOR INFORMATION Corresponding Author * [email protected] (Houyu Wang) * [email protected] (Yao He)



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Dual-Amplification Strategy-Based SERS Chip for Sensitive and

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