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Detection of Circulating Tumor DNA in Human Blood via DNA-Mediated Surface-Enhanced Raman Spectroscopy of Single-Walled Carbon Nanotubes Qifeng Zhou, Jing Zheng, Zhihe Qing, Mengjie Zheng, Jinfeng Yang, Sheng Yang, Le Ying, and Ronghua Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00108 • Publication Date (Web): 30 Mar 2016 Downloaded from http://pubs.acs.org on April 2, 2016

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Analytical Chemistry

Detection of Circulating Tumor DNA in Human Blood via DNA-Mediated Surface-Enhanced Raman Spectroscopy of Single-Walled Carbon Nanotubes Qifeng Zhou†, Jing Zheng†,*, Zhihe Qing‡, Mengjie Zheng† , Jinfeng Yang†, Sheng Yang‡ , Le Ying†, Ronghua Yang‡,*

†

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and

Chemical Engineering, Hunan University, Changsha, 410082, China; ‡ School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha, 410004, China.

*To whom correspondence should be addressed:

E-mail: [email protected]; [email protected] Fax: +86-731-8882 2523

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ACS Paragon Plus Environment


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ABSTRACT: The levels of circulating tumor DNA (ctDNA) in the peripheral blood have been associated with tumor burden and malignant progression. However, ultrasensitive detection of ctDNA in blood remains to be explored. Herein, we developed a new approach, employing DNA-mediated surface-enhanced Raman scattering (SERS) of single-walled carbon nanotubes (SWNTs), that allowed ultrasensitive detection of a broad range of ctDNAs in human blood. Combined with the efficient ctDNA recognition capacity of our designed triple-helix molecular switch and RNase HII enzyme-assisted amplification, the T-rich DNA-mediated SERS enhancement of SWNTs could read out the content of KRAS G12DM as low as 0.3 fM, with the detection of 5.0 µL sample volume, which has potential for point-of-care testing (POCT) in clinical analysis.

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INTRODUCTION Circulating DNA fragments carrying tumor-specific sequence alterations (circulating tumor DNA, ctDNA) are found in the cell-free fraction of blood.1-2 CtDNA, as one of effective disease-related biomarkers present as single-stranded or double-stranded in peripheral blood, usually represents a variable and generally small fraction of the total circulating DNA. 3-4 Studies have shown that ctDNA levels reflect the total systemic tumor burden, in that ctDNA levels decreased upon complete surgery and generally increased as new lesions became apparent upon radiological examination.5 Since ctDNA is defined by mutations and other genomic changes that are indicators of cancer cells, false positive is comparatively rarer in liquid biospy.6 Moreover, ctDNAs, especially for single-stranded ctDNA, have a short half-life which is less than two hours in blood while most protein biomarkers could stay for several weeks, making it can give a much clearer view of the tumor’s present rather than its past.7-8 Due to these unique properties, ctDNAs assay in blood could serve as an effective tool for liquid biopsy, potentially replacing other biomarkers in certain diagnostic applications. The classical methods for monitoring ctDNAs in blood mainly include the DNA sequencing and polymerase chain reaction (PCR).7,9-10 Although these technologies have led to significant contributions to ctDNA detection, the further application still hindered by the complex sample preparation and the interference caused from the constituents of biological environment. For instance, Sim et al proposed a peptide nucleic acid (PNA)-based nanoplasmonic biosensor for detection of tumor-specific genetic and epigenetic markers of ctDNA of PIK3CA gene. 11 Recently, Kelley et al reported an electrochemical clamp assay that could directly detect mutated circulating nucleic acids in patient serum. 12 It is the first successful detection of cell-free nucleic acids, although complicated fabrication of clamp assay chip and unstable electrochemical signal output would provide a lot of space for the follow-up development.13

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In this paper, we describe a new SERS-based strategy via DNA-mediated SERS enhancement of single-walled carbon nanotubes (SWNTs), which can realize detection of single-stranded ctDNA in human blood. To realize high specific identification of target ctDNA, the triple-helix molecular switch (THMS) structure was chose as the recognition element.14-15 In TMHS structure, a thymine-rich single-stranded DNA (T-rich ssDNA) was employed as signal transduction probe while a RNA-site was embedded in the capture sequence to produce numerous T-rich ssDNA in the presence of target ctDNA with aid of RNase HII. Due to the π-π stacking interactions between ssDNA and SWNTs,16-17 the released T-rich ssDNA of THMS could be noncovalently absorbed onto the surface of SWNTs to form the SWNT/ssDNA complex. Because of the specific binding affinity between T base and copper ions (Cu2+),18-20 the SWNT/ssDNA complex then served as templates for the in situ growth of copper nanoparticles (CuNPs) on the surface of SWNT. Since there is an electromagnetic enhancement effect associated with large local E-field between CuNPs and SWNT, 21 obvious SERS enhancement of SWNT including radial breathing mode (RBM) and tangential mode (G-band) could be observed. Interestingly, we found this CuNPs-enhanced SERS effect of SWNT demonstrated an excellent a T-rich ssDNA-concentration dependent pattern which provides a promising platform for target analysis. Owing to the peaks of radial breathing mode (RBM) and tangential mode (G-band) of SWNTs are sharp and obvious that can be easily distinguished from fluorescence backgrounds,22-25 this T-rich ssDNA-mediated SERS enhancement of SWNTs could thus readout the content of ctDNA in complex samples. Compared to the previous reported ctDNA sensing strategy, our design possesses some remarkable features: First of all, only one initiator is required to trigger numerous ssDNA releasing with aid of RNase HII, meanwhile, we just need to change different label-free capture probe when different ctDNAs present. Thus, ultrahigh sensitivity and good versatility could be achieved for multiple ctDNA detection with one SERS-active substrate. Secondly,

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due to the released T-rich ssDNA could serve as specific templates for the in situ growth of CuNPs on the surface of SWNTs to enhance the SERS effect, the false positive signal caused by the other sequences could be reduced effectively. Thirdly, the sharp, back-ground-free Raman signatures of SWNTs can enable us to apply this new approach in complex biological environment. For all we know, it is the first time to detect ctDNA by using SERS assay, which will provide a promising tool for highly sensitive target analysis and clinic noninvasive liquid biopsy. EXPERIMENTAL SECTION Materials and Apparatus. All oligonucleotides used in this experiment were synthesized by TaKaRa Biotechnology Co. Ltd. (Dalian, China). All chemicals were used as received, unless otherwise stated. Human serum, colon cancer tissue and normal colon tissue were provided by Hunan Provincial Tumor Hospital, Central South University (Changsha, China). SWNTs were purchased from Carbon Nanotechnologies, Inc. (Texas, USA). Ultrapure water obtained from a Millipore water purification system (18 M Ω) was used in all assays. 50 nm thick Au films, provided by Mloptic Ltd.(Nanjing, China) were deposited on a 1 nm thick Cr adhesive layer coated onto 20×20×1 mm BK7 glass slides (n = 1.516). SERS measurements were performed using a confocal microprobe Raman instrument (Ram Lab-3010, Horiba Jobin Yvon, France), and spectra were acquired using an 632.8 nm He-Ne laser and 50×long working objective lens (8 mm). CuNPs used in this work were characterized by atomic force microscopy (AFM) were performed on a Bioscope system (Brucker, Inc.). The size and shape of Raman substrate under different conditions were determined by transmission electron microscope (TEM) and scanning electron microscope (SEM) images obtained on JEM-100CXII microscope and JSM-6700F microscopes (JEOL, Ltd., Japan), UV-Vis absorption spectra were recorded on a Hitachi U-4100 UV/Vis spectrophotometer (Kyoto, Japan), and Zeta potential obtained on Zetasizer Nano ZS90(Malvern Instruments)

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respectively. Formation

of

Single-Stranded

DNA-Mediated

SWNT@CuNPs.

Noncovalent

functionalization of SWNTs with single-stranded DNA (ssDNA: A20, T20, C20, G20 and R20) have been well established in earlier reports.16-17 The SWNT/ssDNA suspension was centrifuged at 14000 rpm for 1 h to remove aggregates. The SWNT/ssDNA mixture solution was diluted to 1.0 mL with MOPS buffer (10 mM MOPS, 150 mM NaCl, pH=7.6) and aliquot of stock solution of copper sulphate (CuSO4) was transferred into it. The solution was then incubated at 20 oC for 15 min to form the SWNTs/ssDNA/Cu 2+ complex, following by centrifuged at 14000 rpm for 1 h and then washed with MOPS buffer for three times to remove free CuSO4, and then stored at 4 °C for further usage. To synthesize ssDNA-mediated SWNT@CuNPs, aliquot of 10 μL of the SWNT/ssDNA/Cu 2+ mixture solution was added to a 500 μL volumetric pipe with MOPS buffer. Then, 10 μL sodium acetate (Na 3C6H5O7) (2 mM) was added to the solution with vigorously vortex. After incubated for 100 min at room temperature, the ssDNA-mediated SWNT@CuNPs were formed. Conjugation of THMS on the Surface of Au Film. The Au film was cleaned before surface modification, then, 20 μL of 100 μM T-rich ssDNA were incubated with Au film for 18 h, 20 μL 0.1 M NaCl solution was then added subsequently (pH 6.0, 10 mM MOPS buffer) for aging another 36 h at 4 °C. Finally, Au film was washed by 0.1 M NaCl solution to remove free T-rich ssDNA (1.92×1014/cm2). After conjugation of T-rich ssDNA on Au film, DNA capture probe was added onto the surface of Au film to form THMS under pH 5.0, 10 mM MOPS. Then, Au film was washed by MOPS buffer to remove free capture probes. The absorption maximums (260 nm) of the supernatant, containing free capture probes removed from the Au film, were converted to molar concentrations of DNA by UV absorption spectrometry using published sequence-dependent absorption coefficients. Finally, the average efficiency of successfully conjugated THMS on the surface of Au film was obtained

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(8.32×1013/cm2). ctDNA Extraction from Cancer Tissues and Blood. Tumor specimens were collected after colon surgery from donors of Hunan Provincial Tumor Hospital, Central South University, fixed in formalin and embedded in paraffin. The dissected tumor tissue was digested overnight at 60 °C in 15 µL ATL buffer (Qiagen) and 10 µL proteinase K (20 mg/ml; Invitrogen). DNA was isolated using the QIAamp DNA Micro Kit (Qiagen) following the manufacturer’s protocol. The procedure described briefly as following: firstly, 500 μL lysis/binding solution was added and vortex vigorously to completely lyse the tissues to obtain a homogenous lysate. Secondly, added 1/10 volume of DNA homogenate additive and then incubated 10 min on ice. After that, a volume of acid-phenol: chloroform that is equal to the lysate volume was added before addition of the DNA homogenate additive, following by vortex for 60 s to mix, and centrifuged for 5 min at maximum speed (10,000 ×g) at room temperature to separate the aqueous and organic phases. Finally, dry out the enriched DNA and store at -20 ℃. All plasma samples were centrifuged once for 5 min at low speed (600 ×g) and the resulting plasma sample was spun once more for 10 min at high speed (2000 ×g) in order to free plasma from any remaining blood cells. And the follow isolation procedures ar e the same with tissues. RESULTS AND DISCUSSION Figure 1 shows schematic illustrations of direct detection of single-stranded ctDNA in human blood via DNA-mediated SERS enhancement of SWNTs. The whole SERS sensing platform consisted three key elements: 1) With the advantages of separating molecular recognition and signal transduction, the designed THMS was proposed as the recognition and signal amplification unit.14-15 To facilitate the RNase HII-assisted amplification, a RNA site was embedded in the central sequence of the capture probe. Since RNase HII can recognize and catalyze the hydrolysis of phosphodiester bond 5’ to the ribonucleotide at the perfect

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DNA-RNA junction, numerous signal transduction probes would be generated following the addition of target single-stranded ctDNA with the aid of RNase HII; 26-27 2) Considering the triple helical stem region of THMS is mainly based on Watson-Crick and Hoogsteen base pairings such as T-A•T and C-G•C,28-29 the signal transduction probe was designed as T-rich DNA sequences; 3) Due to the high affinity between thymine and Cu 2+,18-20 and the π-π stacking interactions between single-stranded DNA and SWNTs,16-17 the released T-rich single-stranded DNA of THMS could serve as specific templates for the in situ growth of CuNPs on the surface of SWNTs. Specifically, we found the CuNPs decoration on SWNTs surface could greatly enhance the G-band peak of SWNTs due to the electromagnetic enhancement effect with ssDNA-concentration dependent pattern. Due to the sharp and obvious G-band peaks of SWNTs can be easily distinguished from fluorescence backgrounds,10-11 this new signal output combing with RNase HII-assisted amplification is thus suitable for ctDNA sensing in human blood. To demonstrate the feasibility of this T-rich ssDNA-meditated SERS enhancement of SWNTs, we investigated the T20 sequences (Table S1) serving as templates for the in situ growth of CuNPs on the surface of SWNTs. To compare, another four kinds of 20 bases ssDNA (A20, C20, G20 and R20, Table S1) were employed as controls. To fabricate SERS-active high sensitive substrates, ssDNA-mediated SWNT@CuNPs were synthesized in the solution phase using in situ Cu2+ attachment and seeded growth methods. Raw SWNTs were first modified by ssDNA through noncovalent binding. The decreasing ζ-potential for SWNT/ssDNA indicated the successful ssDNA binding on SWNT. Subsequently, excess CuSO4 was added to form SWNT/T20/Cu2+ complex following by centrifugation at 14000 rpm for 1 h to remove free Cu 2+, the ζ- potential was measured to be -18.2 mV(Figure S1). After reducing the SWNT/T20/Cu2+ complex with sodium citrate (Na3 C6H 5O7), CuNPs were formed on the surface of SWNT. UV-Vis spectra of SWNT@CuNPs firstly displayed the

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absorption peaks around 320 nm, which was attributed to the surface plasmon resonance of synthesized CuNPs (Figure S2). Furthermore, as shown in Figure 2A, the atomic force microscope (AFM) image demonstrated that upon addition of CuSO 4 and sodium citrate to the SWNT/T20 complex solution, many bright purple spots appeared with a mean height about 4.0 nm, indicating the successful growth of CuNPs on the surface of SWNTs. On the contrary, the average height of bare SWNTs was about 2.0 nm (Figure S3A). Meanwhile, the observed Raman bands centered at D-band (1348 cm-1 ) and strong G-band (1605 cm-1) of SWNTs (Figure 2B). Remarkably, most significant SERS signal enhancement of SWNTs could be observed for the T20-mediated SWNT@CuNPs nanocomposites (Inset of Figure 2B). To further comprehend the mechanism of T20-mediated SERS performance of SWNTs@CuNPs, a

theoretical simulation was executed utilizing finite-difference

time-domain (FDTD) to reveal the relationship between the electric magnetic (EM) field and surface density of the growth of CuNPs on core SWNTs. 30-31 As shown in Figure 2C, Au film was chosen as an effective separation and SERS enhancement substrate, 15 which indicated that

strong

plasmonic

coupling

core-satellite(SWNTs@CuNPs@Au-film)

can

be

acquired

nanocomposite.

by

Additionally,

fabricating the

EM-field

enhancements of these nanocomposites were calculated. Specifically, the EM-field enhancements of SWNTs@CuNPs exhibited up to about 69.2-fold which was much higher than that without CuNPs decoration (about 15.5-fold). Since the Raman intensity was proportional to the fourth power of the electric field (|E|4), and |E|4 can be served as an upper limit to experimental calculations of the SERS electric field enhancement,21 therefore, the FDTD data essentially demonstrated the same trend as the experimental result shown in Figure 2B and could verify the mechanism of T20-mediated SERS enhancement effect of [email protected] As the important signal transduction element, we then explored the potential of this T20-mediated SERS enhancement of SWNTs for the substantial quantitative

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analysis and measured the SERS spectra of SWNT@CuNPs as functions of different concentrations of T20. Figure 2D revealed that the SERS signal significantly enhanced upon increasing T20 concentration under the optimized condition (optimized CuSO 4 concentration, CuNPs growth time and pH were shown in Figure S4, Figure S5 and Figure S6). The value of I/I0, where I0 and I are the SERS intensities at 1605 cm -1 in the absence and the presence of T20, linearly increased with the T20 concentration between 0.01 and 20 nM. To realize detection of a specific ctDNA target, we constructed an unique THMS structure as the recognition unit on the surface of Au film and the point mutations of single stranded KRAS G12DM (c.35G>A) and KRAS G12DN were chose as targets in our study (In subsequent experiment, we named them T1 and T2, respectively). 34 The T-rich ssDNA were designed as the signal transduction probes of THMS and the recognition portions were composed of complementary sequences for T1 (capture probes, CP1-4, Table S1). We then characterized each step in the construction of T-rich ssDNA-mediated SWNTs@CuNPs on the surface of Au film. As shown in Figure S7, the scanning electron microscope (SEM) images and corresponding energy-dispersive X-ray spectroscopy (EDS) demonstrated that the SWNTs were immobilized on the surface of T-rich ssDNA-functionalized Au film and many bright purple spots then appeared indicating CuNPs were synthesized. Then, we measured the SERS spectra triggered by T-rich ssDNA released from the THMS structure upon T1 addition. As shown in Figure 3A, about 675.48 %-fold SERS signal enhancement could be observed in the presence of 10 nM T1 and we could achieve ~1.0 pM detection limit based on 3σb/slope (as shown in curve a, Figure 3B), where σ b was the standard deviation of blank samples, under optimized conditions (the optimized capture probe sequence was shown in Figure S8). Considering the low abundance of ctDNA (about 1810 to 12639 fragments/mL) in human plasma,5 we reasoned that whether the nucleic acid enzyme such as RNase HII could be employed to facilitate amplify T-rich ssDNA concentration and thus improve sensitivity.

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As shown in Figure S9, the agarose gel electrophoresis result demonstrated that when incubated CP2/T1 hybrid duplex together with RNase HII, the complex was cleaved to a short bp length while the faint band was ascribed to the undigested segments, which effectively confirmed the RNase HII-catalyzed cleavage mechanism. After repeated replication cycles with aid of RNase HII, the net signal gain produced by this approach was significantly enhanced and we could observe about 4914.15% fold increase upon 10 nM T1 addition (Figure 3A), indicating that RNase HII-assisted amplification was great enough to produce obvious SERS signal change in quantitative analysis. With optimized RNase HII incubation time and RNase HII amount (Figure S10 and Figure S11), a dramatic SERS enhancement was observed in the concentration ranging from 10 fM to 1.0 nM and the limit of detection was ~0.3 fM (as shown in curve b, Figure 3B). Additionally, Figure 3C displayed the Raman mapping images of THMS-functionalized Au film after exposed to different concentrations of T1 ranging from 1 pM to 1 nM. The images were generated by plotting the integrated intensity of 1605 cm-1 peak at each point (10 m x 10 m pixel size). In the maps, uniform SWNT signals were observed within THMS-conjugated spots exposed to T1 at concentrations higher than about 10 fM. At lower concentrations, the SWNTs signal were sparse, which were consistent with a small number of T1, indicating the quantitative detection capability of this SERS assay. The specificity of this strategy mainly depend on the fidelity of RNase HII which only can recognize and cleave the perfect matched RNA/DNA hybrid substrate. 35 To evaluate the specificity of this strategy, T1 and T2 were mixed together in a total concentration of 10 nM while the molar ratios were set as 1000:1, 500:1, 200:1, 50:1 and 10:1, respectively. The results were displayed in Figure S12 and could demonstrate the excellent capability in sensing low abundance of mutation among a great quantity of wild types. Since the release of ctDNA into the blood is thought to be related to the apoptosis and necrosis of cancer cells in the tumour tissues,3 we firstly applied this assay on crude extracts

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of colon carcinoma tissues and para-carcinoma tissues (less than 2 cm close to carcinoma, which containing both normal and cancerous tissues). It is worth noting that the ctDNA extraction we attained from tissues and serum both contained double-stranded and single-stranded ctDNA. According to the previous report, the size of double-stranded ctDNA varies between 70 to 200 base pairs,3 thus, the competitive hybridization between the capture probe (about 15 bases) and the double-stranded ctDNA containing in DNA extraction was hindered by the strong basing paring force of the double-stranded ctDNA itself. Specifically, it was not necessary for us to isolate single-stranded ctDNA form the extraction because the double-stranded ones would not generate obvious signal upon washing treatment. As shown in Figure S13, the colon carcinoma tissues demonstrated strong SERS signals. As a negative control, the signal obtained from para-carcinoma tissues was significantly lower than that from colon carcinoma tissues. As shown in Figure 4A, the intensity of normal tissues was nearly as the control and almost invariable while the value of I/I0 (where I0 and I are the SERS intensities at 1605 cm-1) of carcinoma tissues obtained from 6 representative cases was significantly higher than that obtained from the para-carcinoma tissues, which was in excellent agreement with the clinic results. Therefore, we have proven that this method can distinguish cancer and non-cancer tissues selectively, demonstrating its potential for colon cancer early diagnosis. Many of the tumors are only accessible through fine-needle aspirates, especially for solid tumor such as colon carcinoma and breast carcinoma, with insufficient material available for genotyping. On the contrary, blood can be easily obtained from patients during routine outpatient visits while it is simple and convenient to prepare and store plasma and serum. 1 Therefore, liquid biopsy, especially for the point-of-care testing (POCT) based on the assessment of patient blood samples for mutant ctDNA, is a promising approach. In this research, we explored the feasibility of this strategy for direct detection of single-stranded

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KRAS G12DM in blood since recent researches have reported that the expression levels of KRAS G12DM could provide a new diagnostic biomarker for colon carcinoma. 36 As shown in Figure S14 and Figure S15, the SERS intensity measured from serum sample of a colon carcinoma patient is substantially higher than that from a healthy donor. Such a difference suggests that KRAS G12DM has a high expression level in the colorectal cancer patient blood. To demonstrate the capability of POCT, we challenged our assay with direct single-stranded ctDNA detection in a few microliters of serum. Although further optimization steps can most probably lead to even higher selectivity and sensitivity, our SERS-based assays demonstrated the feasibility of a direct homogeneous ctDNA detection in serum. Figure 4B demonstrated the value of I/I0 linearly increased with the volume of human blood between 10 uL and 1 mL and low to 5 uL could produce obvious SERS signal enhancement of SWNT, which can satisfy the need of POCT well. The concentrations of single-stranded KRAS G12DM in serum were further calculated according to the calibration curve in Figure 3B. As shown in Table 1, the median concentration of serum single-stranded KRAS G12DM from colon carcinoma patients (6222±427 fragments/mL) is significantly higher than that of serum KRAS G12DM from 6 healthy persons (668±69 fragments/mL), which is consistent with that the mutation of KRAS G12D means the dysfunction of serving as a tumor-suppressor to inhibit the progression and metastasis of colon carcinoma. 37 To further confirm the accuracy of this SERS assay, the results obtained are in good agreement with those obtained by a microsphere-based rolling circle amplification assay (RCA, experimental details was shown in Supporting Information, representative fluorescence spectra of RCA was shown in Figure S16, the comparison results attained from RCA and our SERS assay was shown in Table S2) These results demonstrated that this strategy could sense ctDNA in blood with high sensitivity, thus possessed a huge potential for applications in the clinic diagnosis of colon carcinoma. To further manifest the generality of our approach, we used the same principle and

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molecular mechanism to design a THMS targeting PIK3CA E542KM (c.1624G>A). High frequency of mutations of the PIK3CA gene in breast cancer endows it a remarkable biomarker, thus, the expression level in blood has been already used to evaluate the progression of breast cancer.4 The capture probes were composed of complementary sequences for PIK3CA E542KM (c.1624G>A). CP6 was chosen as a preferable capture probe according to the previous report to attain best SERS response sensitivity for the subsequent experiment (Figure S17). As expected, in the absence of single-stranded PIK3CA E542KM (we named it T3 in this experiment), T-rich ssDNA could not be released from the THMS structure, thus preventing CuNPs decoration on the surface of SWNT. However, upon the addition of T3 to the solution containing THMS, the strong interaction between CP6 and T3 caused the disassembly of the THMS structure. This allowed massive T -rich ssDNA releasing and then served as templates for the in situ growth of CuNPs with aid of RNase HII, and thus an obvious SERS signal could be observed (Figure S18). Figure S19 demonstrated the SERS signal was proportional to the concentrations of T3 ranging from 10 fM to 1 nM, with a LOD of 1.5 fM, establishing the quantitative detection capability of this SERS detector. To further evaluate the specificity of our assay targeting T3, Figure S20 demonstrated the extraordinary capability of our RNase HII-assisted SERS assay in the detection of a low-abundance mutation among a large quantity of the wild-types. All these results unequivocally support the feasibility and versatility of this assay for multiplex ctDNA detection. Conclusion. In summary, we have proposed a new strategy, for the first time, to detect ctDNA in human blood directly via DNA-mediated SERS of SWNTs. By using the designed THMS as both molecular recognition and signal amplification units, combining T-rich ssDNA-mediated SERS enhancement of SWNTs, the approach can realize direct direction of various single-stranded ctDNA such as KRAS G12DM and PIK3CA E542KM in human blood. The good performance of this approach was ascribed to (1) with aid of RNase HII, one

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initiator is needed to trigger numerous ssDNA release and thus, ultrahigh sensitivity could be achieved for ctDNA detection and (2) the sharp, back-ground-free Raman signatures of SWNTs can ensure its capability for applying this new approach in complex biological environment such as human blood. Finally, these two unique advantages make this assay has high potential for POCT in clinical analysis. We expect this approach could complement current invasive biopsy approaches for cancer early diagnosis and drug resistance in advanced cancers. Acknowledgment. This work is supported by the National Natural Science Foundation of China (21405038, 21135001, 21575018) and the Fundamental Research Funds for the Central Universities. Supporting Information Available: More experimental details 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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REFERENCES [1] Sorenson, G. D.; Pribish, D. M.; Valone, F. H.; Memoli, V. A.; Bzik, D. J.; Yao, S. L. Cancer Epidemiol. Biomarkers Prev. 1994, 3, 67-71. [2] Vasioukhin, V.; Anker, P.; Maurice, P.; Lyautey, J.; Lederrey, C.; Stroun, M. Br J Haematol. 1994, 86, 774–779. [3] Schwarzenbach, H.; Hoon, D.; Pantel, K. Nat. Rev. Cancer. 2011, 11, 426-437. [4] Gormally, E.; Caboux, E.; Vineis, P.; Hainaut, P. Mutat. Res. 2007, 635, 105-117. [5] Diehl, F.; Schmidt, K.; Choit, M.; Kinzler, K.; Vogelstein, B. Nat. Med. 2008, 14, 985-990. [6] Yong, E. Nature 2014, 511, 524-526. [7] Bettegowda, C.; Sausen, M.; Leary, R. J.; Kinde, I.; Velculescu, V. E.; Kinzler, K. W.; Vogelstein, B.; Papadopoulos, N.; Diaz, L. A. Sci Transl Med. 2014, 6, 224ra24. [8] Dawson, S. J.; Rosenfeld, N.; Caldas, C. N. Engl. J. Med. 2013, 369, 93-94. [9] Murtaza, M.; Dawson, S. J.; Tsui, D. W.; Gale, D.; Forshew, T.; Piskorz, A. M.; Rosenfeld, N. Nature 2013, 497, 108-112. [10] Newman, A.M.; Bratman, S.V.; To J.; Wynne, J.F.; Eclov, N.C.; Modlin, L.A.; Liu, C.L.; Neal, J.W.; Wakelee, H.A.; Merritt, R.E.; Shrager, J.B.; Loo, B.W. Jr; Alizadeh, A.A.; Diehn, M. Nat Med. 2014, 20, 548-54. [11] Nguyen, A.H.; Sim, S.J. Biosens Bioelectron. 2015, 67, 443-449. [12] Das, J.; Ivanov, I.; Montermini, L.; Rak, J.; Sargent, E.; Kelley, S. Nature Chem. 2015, 7, 569-575. [13] Bakker, E.; Qin, Y. Anal. Chem. 2006, 78, 3965. [14] Zheng, J.; Li, J.; Jiang, Y.; Jin, J.; Yang, R.; Tan, W. H. Anal. Chem. 2011, 83, 6586-6592. [15] Zheng, J.; Jiao, A.; Yang, R.; Ma, C.; Jiang, Y.; Deng, L.; Tan, W. H. J. Am. Chem. Soc. 2012, 134, 19957-19960. [16] Yang, R. H.; Jin, J.; Chen, Y.; Shao, N.; Kang, K.; Zhu, Z.; Tan, W. H. J. Am. Chem. Soc.

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Page 17 of 25

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2008, 130, 8351-8358. [17] Zheng, J.; Bai, J. H.; Zhou, Q. F.; Li, J. S.; Li, Y. H.; Yang, J. F.; Yang, R. H. Chem. Commun., 2015, 51, 6552-6555. [18] Rotaru, A.; Dutta, S.; Jentzsch, E.; Gothelf, K.; Mokhir, A. Angew. Chem. Int. Ed. 2010, 49, 5665-5667. [19] Qing, Z. H.; He, X. X.; He, D. G.; Wang, K. M.; Xu, F. Z.; Qing, T. P. Angew. Chem. Int. Ed. 2013, 52, 9719-9722. [20] Li, J. J.; Zhu, J. J.; Xu, K. Trend. Anal. Chem. 2014, 58, 90-98. [21] Zhu, Y.; Jiang, X.; Wang, H.; Wang, S.; Wang, H.; Sun, B.; Su, Y.; He, Y. Anal Chem. 2015, 87, 6631-6638. [22] Rao, A. M.; Richter, E.; Bandow, S.; Chase, B.; Eklund, P. C.; Williams, K. A.; Dresselhaus, M. S. Science 1997, 275,187-191. [23] Chen, Z.; Tabakman, S. M.; Goodwin, A. P.; Kattah, M. G.; Daranciang, D.; Fan, S.; Dai, H. Nat. Biotechnol. 2008, 26, 1285-1292. [24] Chu, H.; Wang, J.; Ding, L.; Yuan, D.; Zhang, Y.; Liu, J.; Li, Y. J. Am. Chem. Soc. 2009, 131, 14310-14316. [25] Wang, X. J.; Wang, C.; Cheng, L.; Lee, S. T.; Liu, Z. J. Am. Chem. Soc. 2012, 134, 7414-7422. [26] Gerasimova, Y. V.; Peck, S.; Kolpashchikov, D. M. Chem. Commun., 2010, 46, 8761-8763. [27] Rydberg, B.; Game, J. Proc. Natl. Acad. Sci. USA. 2002, 99, 16654-16659. [28] Chen, Y.; Lee, S. H.; Mao, C. D. Angew. Chem. Int. Ed. 2004, 43, 5335-5338. [29] Grossmann, T. N.; Roglin, L.; Seitz, O. Angew. Chem. Int. Ed. 2007, 46, 5223-5225. [30] Mubeen, S.; Zhang, S. P.; Kim, N.; Lee, S.; Kramer, S.; Xu, H. X.; Moskovits, M. Nano Lett. 2012, 12, 2088-2094. [31] Tian, C. F.; Ding, C. H.; Liu, S. Y.; Yang, S. C.; Song, X. P.; Ding, B. J.; Li, Z. Y.; Fang, J. 17



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X. ACS Nano 2011, 5, 9442-9449. [32] Moskovits, M. J. Raman Spectrosc. 2005, 36, 485-496. [33] Theiss, J.; Pavaskar, P.; Echternach, P. M.; Muller, R. E.; Cronin, S. B. Nano Lett. 2010, 10, 2749-2754. [34] De Roock, W.; Biesmans, B.; De Schutter, J.; Tejpar, S. Mol. Diagn. Ther. 2009, 13, 103-114. [35] Donis-Keller, H. Nucleic Acids Res. 1979, 7, 179-192. [36] Diehl, F.; Li, M.; He, Y.; Kinzler, K.; Vogelstein, B.; Dressman, D. Nat. Meth. 2006, 3, 551-559. [37] Knickelbein, K.; Zhang, L. Genes Dis. 2015, 2, 4-1

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Figure Captions Figure 1. (A) Construction of SWNTs-based SERS assay coupling with RNase HII-assisted amplification for highly sensitive detection of ctDNA in human blood. Enlarged image illustrates T-rich DNA-mediated growth of CuNPs to enhance the SERS signal of SWNTs. (B) The mechanism of RNase HII-assisted THMS-based amplified recognition to produce numerous T-rich ssDNA. Figure 2. (A) Representative AFM image of T20-mediated growth of CuNPs on SWNT surface. (B) SERS spectra of SWNT and DNA-mediated SWNT@CuNPs with A20, T20, C20, G20, random DNA. [CuSO 4]=150 [Na3C6H5O7]=2 mM. The concentration of SWNT were excessive. (C) FDTD simulation of the normalized EM-field intensity distribution (|E|2/|E0 |2) for T20-mediated SWNT@CuNPs. (a) XZ plane views of T20-mediated SWNT and (b) XZ plane views of T20-mediated SWNT@CuNPs on the surface of Au film. D) SERS intensity enhancements of the 1605 cm -1 -band of SWNT, I/I0, plotted against the concentration of T20. Figure 3. Performances of SERS detection of ctDNA. (A) SERS spectra of SWNTs before (black) and upon 10 nM T1 addition without (blue) and with (red) RNase HII-aided amplification. (B) SERS intensity enhancements of the 1605 cm -1 -band of SWNT, I/I0 , plotted against the concentration of T1 without (a) and with (b) RNase HII-aided amplification. (C) Raman mapping images showing the enhanced the 1605 cm-1 peak of SWNT with different concentrations of T1 in PBS coupling with RNase HII-aided amplification. [CP2]=400nM, [CuSO4]=150 [Na3C6H5O7]=2mM. The concentration of SWNT was excessive. Figure 4. (A) Histogram showing the results from 6 patients (a-f) with colon cancer. Blue bar

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represents carcinoma tissue and grass green bar represents para-carcinoma tissue. (B) SERS intensity enhancements of the 1605 cm -1 -band of SWNT@CuNPs, I/I0 , plotted against the volume of human blood with(a) and without(b) RNase HII-aided amplification. [CP2]=400 nM, [CuSO4]=150  [Na3C6H5O7]=2 mM. The concentration of SWNTs were excessive. Table 1. SERS assays of serum samples for KRAS G12DM detection.

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Figure 1

21



3

b

2 1 0

Z/nm

-6

-4

-2

T20 A20 C20 G20 R20

5000 1348

SWNTs

4

6 4

45

3

6.0

18

C20 R20 G20 A20

4.5

0

3.0 1.5

9

0.0 0.0 0.5 1.0 1.5 2.0 [T20]/nM

0

1350 1500 1650 -1 Raman shift/cm

2

3

0 1200

0

X/nm

27

18 9

10000

4

36

T20

27

a

I/I0

15000

1605 36

I/I0

20000

(D)

45

1800

2

|E| /|E | 0 5

I/I0

(B) 25000

Z/nm

Z/nm

(C)

-1 0 1 2 3 4 -1 0 1 2 3 4

2

(A)

SERS signal(arb, units)

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0

40

80 120 [T20]/nM

Figure 2

22


160

200

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(B)

1605

20000

40

b

32

30

15000 4914.15%

10000

20

24

I/I0

I/I0

(A) SERS signal(arb, units)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


b 10

16

a 0

5000

8

a

675.48%

0

10fM 100fM 1pM 10pM 100pM 1nM

[T1]

0

1350

(C)

Blank

1500 1650 -1 Raman shift/cm

1.0 pM

1800

0

2

4

6

[T1]/nM

10 pM

100 pM

1.0 nM

Figure 3

23


8

10


(A)

para-carcinoma carcinoma

32

(B) 6.0

a

24

I/I0

4.5

I/I0

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16 3.0

b

8 1.5

0

a

b

c

d

e

f

0.0

0.2

0.4

0.6

0.8

[Human blood]/mL

Figure 4

Table 1

24


1.0

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Detection of Circulating Tumor DNA in Human ... - ACS Publications

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