Ultrasensitive Detection of Circulating Tumor DNA of Lung Cancer via

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Biological and Medical Applications of Materials and Interfaces

Ultra-sensitive Detection of Circulating Tumor DNA of Lung Cancer via Enzymatically Amplified SERS Based Frequency Shift Assay Jie Zhang, Yuhang Dong, Wenfeng Zhu, Dan Xie, Yuliang Zhao, Dayong Yang, and Min Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02953 • Publication Date (Web): 03 May 2019 Downloaded from http://pubs.acs.org on May 3, 2019

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ACS Applied Materials & Interfaces

Ultra-sensitive Detection of Circulating Tumor DNA of Lung Cancer via Enzymatically Amplified SERS Based Frequency Shift Assay Jie Zhang#,a,b, Yuhang Dong#,a, Wenfeng Zhub, Dan Xieb, Yuliang Zhaob, Dayong Yang*,a, Min Li*,b aFrontier

Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering

(MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, China. bCAS

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of

High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China

KEYWORDS: circulating tumor DNA (ctDNA); surface-enhanced Raman scattering (SERS); frequency shift; RNase; DNA nanostructures

ABSTRACT: Circulating tumor DNA (ctDNA) is a promising noninvasive biomarker for the early diagnosis of cancers. However, it is challenging for accurate and sensitive detection of pico-to-femtomolar serum concentration of ctDNA, especially in the presence of its analogues which produce strong background noise. Herein, a DNA-rN1-DNA mediated surface-enhanced Raman scattering (SERS) frequency-shift assay is developed, which enables sensitive detection

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of ctDNA with one single base-pair mutation (KARS G12D mutation) from the normal ones (KARS G12D Normal) of lung cancer. This sensing platform features in both the designed hairpin DNA-rN1-DNA probe for specific ctDNA recognition and the employed RNase HII enzyme which specifically hydrolyzes DNA-rN1-DNA/ctDNA hybrid and thus allows ctDNA recycling in the system to realize signal amplification. The detection system shows subfemtomolar level sensitivity in phosphate buffered saline (PBS) solution, and is demonstrated to function well in both fetal bovine serum (FBS) and human physiological media. In particular, the sensitive assay of ctDNA in serum samples from lung cancer patients is achieved, suggesting its high potential applications in clinical settings for early diagnosis and prognosis of lung cancer.

INTRODUCTION

Lung carcinoma is one of the most deadly forms of cancers world-wide, with an estimated 1.6 million deaths according to the newest database from WHO.1 The mortality-toincidence ratio is very high (reaching 87%), mainly due to unpredictable clinical manifestations and its faster development and metastases, especially for the non-small cell lung cancer (NSCLC), as well as the low accuracy of early detection techniques.2 The five-year survival rate of lung cancer is in the range of 10–20% in most countries according to the latest statistics (from 2000-2014).3 If patients are diagnosed and treated at the early stage, the situation can be greatly improved, leading to a > 80% 5-year survival.4 Unfortunately, approximately 75% of lung cancer patients are diagnosed at the advanced stage due to poor diagnostic techniques.5 Developing accurate, sensitive and specific clinical methods for early diagnosis of lung cancer continues to be a clinical challenge.


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Circulating tumor DNA (ctDNA), a subset of cell free DNA (cfDNA) released from the tumor cells to the blood stream, has been considered as a promising biomarker for cancer diagnosis.6-8 ctDNA carries the tumor-specific sequence alterations and thus can be used as a predictor of disease stage and prognosis.9-12 As a new biomarker, ctDNA is an effective target for liquid biopsy which possesses many advantages over tissue biopsy being non-invasive and simple to perform.13 In addition, ctDNA has a quite short half-life which is generally less than 2 hours in blood compared to most protein biomarkers which normally remain for several weeks,6, 14

making ctDNA an appropriate target reflecting the “real time” situation of tumor burden.

Therefore, sensitive and accurate detection of ctDNA has been proven to be significant in tracking the evolution of tumor in real time and serving as a liquid biopsy for clinical diagnosis. However, the typical pico-to-femtomolar concentration of ctDNA in serum as well as the interference from its homologous structures which produce strong background noise, makes ctDNA detection and quantification challenging.9 Nucleic acids play important roles in biosensing techniques due to their precise molecular programmability and abundant “enzymatic toolkits”.15-19 As for ctDNA, signal amplification is the key to facilitating its detection under trace conditions. To achieve this, nucleases were normally introduced to hydrolyze the DNA-ctDNA hybrid, allowing ctDNA to be recycled in a proposed system for signal amplification.20, 21 RNases HII has been demonstrated to possess high specificity and high efficiency to hydrolyze DNA-rN1-DNA/ctDNA hybrid.22, 23 Accordingly, in this work we synthesized a hairpin DNA-rN1-DNA probe with an embedding RNA site (Scheme 1), which can specifically recognize and hybridize with ctDNA. RNase HII directs hydrolysis from the RNA site of DNA-rN1-DNA/ctDNA complex to release ctDNA for the next cycle, leaving the left part, P1, standing on the substrate. In this way, multiple P1 could be produced by


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one ctDNA, achieving the signal amplification. It is noteworthy that RNase HII will not work if DNA-rN1-DNA hybridizes with any other sequences, for example, the analogues to ctDNA in the system (serum), further indicating its high specificity. Surface-enhanced Raman scattering (SERS) has received remarkable attention in recent years due to its broad range of potential applications in disease diagnosis21, 24, 25, environmental surveillance26-28 and food safety supervision29, 30. SERS based sensing strategy possesses distinct advantages such as high spectral resolution, good transparency in biological environments, excellent multiplexing capability and large dynamic range.31 It has been demonstrated that SERS is a promising platform for ctDNA detection.21,

25

For example, Yang et al.21 reported that a

SERS technique could effectively detect ctDNA with a sensitivity of 1.5×10-15 M in solution. Trau et al.25 combined SERS and PCR for ctDNA detection, and achieved sensitivity down to 10 copies. However, the traditional SERS sensing technique, that is, the “sandwich” sensing mode which uses Raman intensity variation as the signal is cumbersome, and especially the fact that multiple reaction steps inevitably leads pronounced nonspecific adsorption which results in decreased specificity and sensitivity. Very recently, a new frequency-shift based SERS method for biomolecules sensing has been developed, which focuses on shifts of the normal mode vibrations of a Raman reporter upon binding of a target.32, 33 Such shifts are usually caused by the structural re-orientation or deformation of the Raman reporter.34 Compared to the traditional “sandwich” SERS assay, the requirement for only one single reaction step is one of the major advantages of the frequency-shift SERS method, which greatly simplifies the detection procedure and, most importantly, largely suppresses nonspecific adsorption and consequently increases its sensitivity and accuracy. In addition, the frequency shift method facilitates the shift measurements in a strong signal rather than distinguishing a peak above noise like the sandwich


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structure mode. We have previously reported a series of works using frequency-shift SERS in view of its pronounced advantages including the simultaneous detection of protein and genetic biomarkers of liver cancer, trace Zn (II) detection in cellular media and glycoprotein assay.27, 33, 35, 36

In the current work, we develop an RNA site embedding DNA-rN1-DNA mediated frequency-shift SERS sensing platform for sensitive detection of ctDNA of lung cancer. A hairpin DNA-rN1-DNA probe was designed for specific ctDNA recognition. RNase HII enzyme was employed to realize signal amplification for ctDNA sensing by specifically cutting DNArN1-DNA/ctDNA hybrid at the RNA site and then releasing ctDNA to the next cycle. Raman spectra of the left P1 sequence on the Raman reporter (5,5’-dithiobis(succinimidyl-2nitrobenzoate, DSNB) after hydrolysis by RNase HII were recorded before and after binding with a foreign sequence (FS) that can specifically hybridize with P1 for ctDNA detection. We show that this method offers high sensitivity (LOD of 1.2×10-16 M [ctDNA]). This system is also shown to function well in human physiological media. Remarkably, this is the first example of sensitive ctDNA detection using the Raman-based frequency shift modality, which may provide a new strategy for highly sensitive analysis of nucleic acids, such as microRNAs.

RESULTS AND DISCUSSION

Scheme 1 shows the schematic strategy of our approach to sensing ctDNA via SERS-based frequency shift method. Our detection platform includes silver nanoparticle film (AgNF) substrates, Raman reporter-DSNB and RNase HII enzyme-assisted amplification. DSNB was chosen in this work as the Raman reporter owing to its large cross section of nitro group at 1334 cm-1, and its sensitive response upon binding of target species based on our previous work.33, 36


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DSNB was chemisorbed on the surface of AgNF through Ag-S bonding, companying with the cleavage of the disulfide bond in DSNB. The SERS spectra of Ag-DSNB were shown in Figure S1. The three dominant bands located at 1078 cm-1, 1334 cm-1 and 1558 cm-1 are corresponded to the

M stretch, the symmetric nitro stretch and an aromatic ring mode (8a).37 The binding of

DNA-rN1-DNA probe occurred via the reaction between primary amine of the probe and the succinimidyl ester of the DSNB. In RNase HII enzyme-assisted amplification system, the hairpin DNA-rN1-DNA probe was designed as the recognition sequence, which could be opened specifically by the target ctDNA as indicated in Scheme 1. On the probe sequence was embedded an RNA site which could be selectively cut by RNase HII in the presence of ctDNA because RNase HII could recognize and catalyze the hydrolysis of the phosphodiester bond /N to the ribonucleotide at the perfect DNA-RNA junction. The cyclic process between the DNA-rN1DNA probe, ctDNA and RNase HII is shown in Scheme 1. After being cut by RNase HII, the left HP part, P1, could specifically capture its complementary sequence FS in solution, resulting in the frequency of the nitro group in DSNB shifting to red. The vibrational frequency shifting to lower energy observed herein could be due to mechanical stretching (reduction in stiffness) of the relevant bonds induced by structural change in the P1 monolayer.36, 38, 39 The Raman spectra were collected before and after FS hybridizing with P1. The typical SEM image of the silver AgNF used for the SERS measurements in this work is shown in Figure 1a. Silver nanoparticles with a diameter of ca. 40 nm were observed to uniformly and densely grow on the glass substrate. Sharp edges of the silver nanoparticles instead of round ones make the AgNF an excellent SERS substrate.

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The corresponding

extinction spectrum of the AgNF before and after decorating with DSNB and successively binding with the hairpin DNA-rN1-DNA probe are presented in Figure 1b. The peak of plasmon


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resonance between particles at around 447 nm for the Ag film shifted to red by around 10 nm after binding DSNB and red shifted by another 4 nm after immobilizing the hairpin DNA-rN1DNA probe. The shift was attributed to refractive index change upon species binding to or leaving the silver film. The reproducibility of Raman signals from AgNFs was assessed for the 1334 cm-1 band of chemisorbed DSNB monolayer at the 780 nm excitation wavelength. Absolute change in Raman shift for the 1334 cmM band was measured in 20 mM PBS after incubating the substrates with RNase HII and T1 concentrations ranging from 10M> to 10M / M and after incubating FS solution. SERS spectra were recorded at three randomly selected positions on each chip for 5 parallel chips prepared from 3 different batches. The SERS response was highly uniform, with relative standard deviations (RSD) less than 3.6% (Figure S3a). Raman mapping showed in Figure S3c also illustrates the high uniformity of the Raman signal from AgNF substrates. In addition, the reproducibility of the change in frequency shifts observed upon FS binding after DNA-rN1-DNA probe cut by RNase HII was also assessed. The change in Raman shift of the

1334 cmM band

was measured after incubating the chips with RNase HII/T1 (T1 concentrations ranging from 10M> to 10M / M) and after the incubation with FS solution (Figure S3b). The averaged standard deviation in the measured shifts over the concentration range and substrates using 780 nm excitation was 0.04 cm-1, same to our previous work36 and slightly higher than the value of 0.022 cm-1 given in the application note for the Raman instrument used here.40 Additionally, considering the experimental cycle of this work is 2 days, the day-to-day reproducibility of the AgNFs chip was assessed. The Ag substrate-DNA platform presents high stability within 3 days as shown in Figure S4. To verify the successful preparation and purification of the hairpin DNA-rN1-DNA probe, a


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similar probe with linear structure (LP) was designed as a reference, which possessed same base composition to the hairpin DNA-rN1-DNA probe (HP) but with different sequences in the last seven bases (Table 1). The last part of sequence in LP was designed to be different from HP, aiming to avoid forming hairpin structure. SYBR Green I, which has stronger affinity to doublestranded DNA (dsDNA) than single-stranded DNA (ssDNA), was employed for HP and LP characterization and comparison. The electrophoresis experiments were firstly performed to prove whether HP could be uncoiled by ctDNA. The results showed that HP (lane 2) and LP (lane 1) presented different mobility, indicating they possessed different structures: hairpin for HP and straight chain for LP (Figure S2a). Meanwhile, the clean single band of HP in lane 2 suggests high purity of our synthesized HP. Two probe structures were further confirmed by the fluorescent assay as shown in Figure S2b. The fluorescence intensity of HP was expectedly higher than that of LP, attributing to the higher affinity of SYBR Green I to dsDNA in the HP hairpin structure. Target ctDNA (T1), KARS G12D mutation, features in only one nucleotide difference compared with the normal sequence (N1: Normal DNA, KARS G12D Normal). After the hybridization with T1, LP/T1 (lane 3) and HP/T1 (lane 4) presented same mobility in electrophoresis analysis, confirming that the hairpin structure of HP was successfully uncoiled by ctDNA. The values of the fluorescence intensity of LP/T1 and HP/T1 were measured to be identical, which further validated the feasibility of our design. To confirm that RNase HII could specifically recognize and hydrolyze the complex of HP/T1, the electrophoresis analysis was carried out (Figure 2a). It was clearly shown that a heavier complex was formed (lane 4), indicating the hybridization of T1 with HP occurred. In the presence of T1 and RNase HII, the band of HP disappeared (lane 6), and two light bands appeared. This suggested that sequences in shorter length were produced in this case, and that


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HP hybridized with T1 completely before hydrolysis by RNase HII to subsequently produce P1 and another sequence with similar length. However, for the case of HP/N1, the band of HP could still be observed, but no new band was observed (lane 5), indicating no interaction occurred between N1 and HP. A very light band between HP and N1 could be distinguished, suggesting that RNase HII could hydrolyze the HP/N1 hybrid slightly. We thus speculated that the specificity of RNase HII was not 100% to HP/T1, which was consistent with the previous report.20 Worthy of note is that the inactive of FS sequence to HP is essential in this work to increase the accuracy of SERS assay. To assess this, the corresponding experiments were designed and performed. Figure 2b clearly showed the band of HP kept at the same position before and after interacting with FS. In contrast, LP/FS complex presented less mobility compared with LP. These results indicated that FS could not hybridize with HP but could interact with LP efficiently. Taken together, the above results confirmed that our DNA design was valid and facilitated the sensitive and specific ctDNA detection via DNA-rN1-DNA mediated frequency-shift sensing strategy. Chloride ion in RNase HII buffer might have effects on AgNF and consequently on the Raman spectra.41,

42

In the assessment experiments, SERS spectra were recorded after the

DSNB/AgNF substrates blocked by butyl amine were immersed in NaCl solutions with various concentrations (Figure S5). The values of PQ Raman shift| changed pronouncedly with time but presented similar modality for the NaCl solutions at different concentrations. The shift changes increased greatly at the beginning, then kept nearly constant for a period of time before final decrease (Figure S5). Additionally, the plateaus of the shift changes were observed to gradually decrease along with the increase of NaCl concentrations. Considering the total concentration of chloride ion in RNase HII buffer was ~30 mM which kept RNase HII most active, we optimized


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30 mM NaCl in buffer as the best experiment condition during the whole measurements. In addition, the experiments on how the PBS buffer with 30 mM NaCl affecting the Raman spectra were performed. The results showed that the peak position of DSNB at around 1334 cm-1 changed dramatically during 0-190 min, then reached a plateau till to 30 h (Figure S6). Parallel experiments showed similar results, which further confirmed the feasibility of controlling the incubation time of P1 with FS between 4-30 h. To ensure that the shift change would not be affected by the existing chloride ion in solution, we kept the pre-incubation time for the substrates in buffer for 1.5 hours, another 1 hour for butyl amine blocking and 1.5 hours for RNase HII enzyme-assisted amplification process in the whole measurements. The optimal conditions for sensing ctDNA included the hairpin DNA-rN1-DNA probe surface density, incubation temperature, incubation time as well as the concentration and the reaction time of RNase HII with HP/T1. The surface density of the hairpin DNA-rN1-DNA probe was vital to get higher Raman signal (shift change). To obtain the optimal surface density of HP on DSNB/AgNF surface (1011 -1013/cm2),43 the DSNB/AgNF substrate was incubated with HP solution at the concentration of 9 M based on our previous work.36 In this case, the number of HP molecules in solution was estimated to be 50 times more than that of the calculated HP on substrate, assuming the formation of a dense self-assembled monolayer of HP molecule with ~2 nm in diameter. The optimal concentration and reaction time of RNase HII to HP/T1 complex are vital to the sensing platform to achieve high performance. The substrates were incubated with 0.2 U/ L RNase HII for different time to obtain the relationship between shift changes in the DSNB nitro stretch and the reaction time. It is reasonable that longer reaction time of RNase HII could lead to higher cutting efficiency as indicated in Figure 3a. A plateau was reached when the incubation time was longer than 90 min, suggesting 90 min could be considered as the


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appropriate reaction time. Similarly, the relationship between shift changes and RNase HII concentrations was shown in Figure 3b, in which the desirable concentration was determined to be 0.2 U/ L. Thus, incubating the substrates with 0.2 U/ L RNase HII at 37 oC for 90 min was selected as the optimal experimental conditions for ctDNA detection. To investigate the performance of this sensor to assay ctDNA in solution, SERS spectra were firstly recorded after incubating the hairpin DNA-rN1-DNA probe bound substrates with ctDNA/RNase HII solutions at 10-8 to 10-16 M T1 concentrations for 1.5 hours. SERS spectra were then recorded after incubating the above substrates with FS solution for 10 hours. With increasing concentrations of T1, the expected changes in the 1334 cm-1 frequency band of the Raman reporter were observed clearly before and after FS interacting with the substrates (Figure 4). Control experiments were performed in the same buffer in the absence of T1 and no observable band shifts were detected. It should be noted that the ability to precisely measure the position of a band is much more a function of the wavelength precision of the instrument than of the spectral resolution. The limiting factor for this analysis is not going to be the resolution, but rather the precision (reproducibility). For the Raman instrument we used in this work, the maximum root-mean-square (RMS) variation in peak position at any single point in the map between the five repeats is only 0.022 cm-1. Considering three times the noise level to definitively distinguish a difference, the instrument can distinguish peak shifts as small as 0.066 cm-1.40 In practice, we found the shot-to-shot variation in peak position to be slightly higher than the value of 0.04 cm-1 for a large number of measurements under various conditions. The shift change greater than 0.15 cm-1 can be considered significant. The semi-log plot of PQ Raman shift| versus T1 concentration was linear as seen in Figure 4c, indicating an exponential relationship, with an LOD of 1.2 × 10-16 M which showing 0.15 cm-1 shift change. These results suggested


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that our designed platform could be used for the quantitative detection of ctDNA in aqueous solution. Efficient distinguishing ctDNA to its analogues is a vital factor to the sensor performance. As a proof-of-principle test, the selectivity of this senor was evaluated in T1 solution with spiked 9 to 10000 folded N1, the most similar analogue (only one base difference), as the interfering substance. The results were shown in Figure 5a. The total concentration of T1 and N1 was 10 nM. The blank (no T) means there is neither T1 nor N1 in solution, and 0 represents 10 nM N1. This sensor could recognize one T1 from 10000 N1 analogues, indicating its high specificity of this sensor in ctDNA detection. The most important performance of a sensor is its potential applications in physiological media. We firstly accessed this in fetal bovine serum (FBS) with spiked T1 after the pretreatment of FBS by AgNO3 to remove the high amount of Cl- and by phenol acid /chloroform to extract the proteins in FBS (see details in Experimental section). The effect of protein extraction on the content of ctDNA in FBS media was assessed and the results were shown in Figure S7. It was clearly seen that the SERS responses presented high reproducibility for different parallel samples after protein extraction in FBS. The RSD before and after extraction was less than 8%, indicating the extraction process did not affect the content of ctDNA in either FBS or serum media. In addition, the results in Figure S7 also presented a >97% recovery of ctDNA in FBS. Figure 5b shows that equimolar FBS solutions with T1 ranging from 10-8 to 10-15 M were observed to have the same trends of downshifts to that in PBS at the band of 1334 cm-1 of DSNB, but with increased uncertainty at 0.9 × 10-14 M, the LOD of the experiments in FBS. This lowest concentration that could be measurable was reduced by two magnitudes than the case in PBS, suggesting the nonspecific interaction and interruption of substances, mainly the nucleic acid


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analogues in FBS to this sensing platform. Finally we tested the sensing capability of this platform in human serum samples. We obtained serum/plasma samples from several patients with primary lung adenocarcinoma (PLA) through the Tianjin Medical University Cancer Institute and Hospital, China. We undertook the frequency shift assay by using the same sensing strategy as above. The results are shown in Table 2. The shift changes in Raman frequency for five patient samples are in the range of 0.19 cm-1 to 0.37 cm-1. These shift values correspond to the ctDNA concentrations of 1.16 to 3.99

10-14 M

10-12 M in serum/plasma by using the frequency shift value versus ctDNA

concentration relationship determined in FBS in Figure 5b as the standard curve. The proof-ofprinciple experiments demonstrate the capability of frequency shift sensing method for trace ctDNA detection in clinical diagnosis. Worthy of note is that no pronounced shift change was observed for nonprimary lung adenocarcinoma as indicated in Table 2, suggesting our sensing strategy could possess the potential of distinguishing between primary and nonprimary lung adenocarcinoma. This will be assessed with extended studies in large cohort in the future.

CONCLUSION

In summary, we have developed an RNA site embedding DNA-rN1-DNA mediated SERS frequency shift sensing platform for the first time for ctDNA assay, which exhibited subfemtomolar level detection sensitivity. RNase HII enzyme was employed for specific hydrolysis of the RNA site of DNA-rN1-DNA/ctDNA complex and recycling ctDNA for signal amplification. This platform could distinguish one base-pair mismatch target ctDNA from 10000 normal ones, indicating its high specificity in ctDNA sensing. The same detection platform applied in FBS showed similar performance but slightly lower LOD (0.9 × 10-14 M), possibly


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and HP at 37 oC. Lane 1: FS; Lane 2: LP; Lane 3: HP; Lane 4 and lane 5 represent LP/FS and HP/FS complex incubated at 37 oC for 1h.

a

b

Figure 3 Effect of conditions of RNase HII on the Raman shift of the 1334 cm-1. a) The Raman shift of the 1334 cm-1 change along with reaction time of RNase HII (0.2 U/ L) with 10-8 M ctDNA; b) The Raman shift of the 1334 cm-1 changes with different concentrations of RNase HII with 10-8 M ctDNA at 60 min. The standard deviations obtained by three repeated measurements are shown as the error bar.


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a

b

c

Figure 4 a) Raman spectra of the detection platform responses to T1 with various concentrations. b) Extended Raman spectra in the region around 1334 cm-1 shows the responses of the detection platform to T1 with various concentrations. c) Semi-log plot of the Raman shift of 1334 cm-1 as a function of T1 concentrations of 10-8, 10-10, 10-12, 10-14, 10-15, 10-16 M. The standard deviations obtained by three repeated measurements are shown as the error bars.


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a

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b

Figure 5 a) Selectivity of this Raman shift ctDNA sensing platform for ctDNA. Histogram showing the capacity to measure the perfect-matched target in a mixture of N1 and T1. The percentage T1 is 0 (no T represent no T1 and N1), 0%, 0.01%, 0.1%, 1%, 10% respectively with a total T1 and N1 concentration of 10-8 M. The standard deviations obtained by three repeated measurements are shown as the error bars. b) Semi-log plot of the Raman shift of 1334 cm-1 as a function of T1 concentrations of 10-8, 10-10, 10-12, 10-14, 10-15 M in FBS. The standard deviations obtained by three repeated measurements are shown as the error bars.


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Table 1 Oligonucleotide sequences used in this study. Strand

Sequence (5’-3’)

Use

NH2-(CH2)6-TCTGTCTTGGAGCTGARa) Probe TGGCGTAG AGACAGA

Combining specifically

target

DNA

TCTGTCTTGGAGCTGAR HP

DNA sequence of Probe TGGCGTAG AGACAGAb) TCTGTCTTGGAGCTGA

LP

HP analogue TGGCGTAG GGAAAACc)

T1

CTACGCCATCAGCTCCA

Target DNA Mutation)

(KARS

G12D

N1

CTACGCCACCAGCTCCA

T1 analogue Normal)

(KARS

G12D

FS

CAGCTCCA AGACAGA

Foreign sequence that specifically combining the left part of probe after being but by RNase HII

a)

AR indicates the RNA site of DNA-rN1-DNA probe that can be specifically recognized by RNase HII after hybridization with T1. b) c) The purple parts of HP and LP highlight the last seven bases possessing same base composition but slightly different base sequences.


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Table 2 Frequency shift assay of ctDNA for human serum/plasma. Samples

Frequency shift change

ctDNA concentration

Patient #1-PLA (serum)

0.20±0.04 cm-1

(1.16~8.86)

10-14 M


0.34±0.03 cm-1

(0.52~2.40)

10-12 M


0.37±0.02 cm-1

(1.45~3.99)

10-12 M

Patient #4-PLA (plasma)

0.22±0.01 cm-1

(4.13~6.87)

10-14 M

Patient #5-PLA (plasma)

0.19±0.01 cm-1

(1.93~3.21)

10-14 M

Patient #6-non-PLA (serum)

-

0


-

0


-

0

Healthy Control #1 (serum)

-

0


-

0


-

0

Note: The short-term “-” indicates that the sample information is undetectable.


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ASSOCIATED CONTENT Supporting Information Supporting Information is available free of charge on the ACS Publications website or from the authors. The Supporting Information involves Experimental section, SERS spectra of the Raman reporterDSNB, SEM image and corresponding extinction spectrum of the AgNF, batch-to-batch reproducibility of Raman intensity and shift, day-to-day reproducibility of Raman intensity and shift, effect of NaCl on the shift change of DSNB/AgNFs, and effect of protein extraction on the content of ctDNA. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] ORCID Dayong Yang: 0000-0002-2634-9281 Min Li: 0000-0003-2959-9080 Author Contributions J.Z. and Y.D. contributed equally to this work. ACKNOWLEDGMENT


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This work was supported in part by National Natural Science Foundation of China (grant no. 21621004, 31671012, 21575101 and 21622404), and the National Basic Research Program of China (2015CB932004). Received: ((will be filled in by the editorial staff)) Revised: ((will be filled in by the editorial staff)) Published online: ((will be filled in by the editorial staff))


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REFERENCES (1) GLOBCAN 2012: Estimated Cancer Incidence, Mortality and Prevalence Worldwide in 2012. http://globocan.iarc.fr/Default.aspx. (2) Arya, S. K.; Bhansali, S. Lung Cancer and Its Early Detection Using Biomarker-Based Biosensors. Chem. Rev. 2011, 111, 6783-6809. (3) Allemani, C.; Matsuda, T.; Di Carlo, V.; Harewood, R.; Matz, M.; Niksic, M.; Bonaventure, A.; Valkov, M.; Johnson, C. J.; Esteve, J.; Ogunbiyi, O. J.; Azevedo e Silva, G.; Chen, W. Q.; Eser, S.; Engholm, G.; Stiller, C. A.; Monnereau, A.; Woods, R. R.; Visser, O.; Lim, G. H.; Aitken, J.; Weir, H. K.; Coleman, M. P.; Grp, C. W. Global Surveillance of Trends in Cancer Survival: Analysis of Individual Records for 37,513,025 Patients Diagnosed with One of 18 Cancers during 2000–2014 from 322 Population-Based Registries in 71 Countries (CONCORD-3). Lancet 2018, 391, 1023-1075. (4) Liu, Y.; Lee, J.; Tsai, D.; Kao, C.; Chang, P.; Chou, S. and Chen, C. Survival of the Fittest: the Role of Video-Assisted Thoracoscopic Surgery in Thoracic Impalement Injuries. J. Thorac. Dis. 2018, 10, 4445-4452. (5) Knight, S. B.; Crosbie, P. A.; Balata, H.; Chudziak , J.; Hussell, T. and Dive, C. Progress and Prospects of Early Detection in Lung Cancer. Open Biol. 2017, 7,170070. (6) Speicher, M. R.; Pantel, K. Tumor Signatures in the Blood. Nat. Biotechnol. 2014, 32, 441443. (7) Newman, A. M.; Bratman, S. V.; To, J.; Wynne, J. F.; Eclov, N. C. W.; Modlin, L. A.; Liu, C. L.; Neal, J. W.; Wakelee, H. A.; Merritt, R. E.; Shrager, J. B.; Loo, B. W.; Alizadeh, J., A. A.; Diehn, M. An Ultrasensitive Method for Quantitating Circulating Tumor DNA with Broad Patient Coverage. Nat. Med. 2014, 20, 548-554. (8) Newman, A. M.; Lovejoy, A. F.; Klass, D. M.; Kurtz, D. M.; Chabon, J. J.; Scherer, F.; Stehr, H.; Liu, C. L.; Bratman, S. V.; Say, C.; Zhou, L.; Carter, J. N.; West, R. B.; Jr, G. W. S. J.; Shrager, B.; Jr, B. W. L.; Neal, J. W.; Wakelee, H. A.; Diehn, M.; Alizadeh, A. A. Integrated Digital Error Suppression for Improved Detection of Circulating Tumor DNA. Nat. Biotechnol. 2016, 34, 547-555. (9) Schwarzenbach, H.; Hoon, D. S. B.; Pantel, K. Cell-Free Nucleic Acids as Biomarkers in Cancer Patients. Nat. Rev. Cancer 2011, 11, 426-437. (10) Diehl, F.; Schmidt, K.; Choti, M. A.; Romans, K.; Goodman, S.; Li, M.; Thornton, K.; Agrawal, N.; Sokoll, L.; Szabo, S. A.; Kinzler, K. W.; Vogelstein, B.; Jr, L. A. D. Circulating Mutant DNA to Assess Tumor Dynamics. Nat. Med. 2008, 14, 985-990. (11) Ignatiadis, M.; Lee, M.; Jeffrey, S. S. Circulating Tumor Cells and Circulating Tumor DNA: Challenges and Opportunities on the Path to Clinical Utility. Clin. Cancer Res. 2015, 21, 4786-4800. (12) Hutchinson, L. CtDNA-Identifying Cancer before It Is Clinically Detectable. Nat. Rev. Clin. Oncol. 2015, 12, 372. (13) Wan, J. C. M.; Massie, C.; Garcia-Corbacho, J.; Mouliere, F.; Brenton, J. D.; Caldas, C.; Pacey, S.; Baird, R.; Rosenfeld, N. Liquid Biopsies Come of Age: Towards Implementation of Circulating Tumour DNA. Nat. Rev. Cancer 2017, 17, 223-238. (14) Dawson, S. J.; Rosenfeld, N.; Caldas, C. Circulating Tumor DNA to Monitor Metastatic Breast Cancer REPLY. New Engl. J. Med. 2013, 369, 93-94. (15) Yang, D., Campolongo, M.J., Tran, T.N.N., Ruiz, R.C.H., Kahn, J.S., Luo, D. Novel DNA


23


Page 24 of 26

Materials and their Applications. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2010, 2, 648-669. (16) Deng, R.; Tang, L.; Tian, Q.; Wang, Y.; Lin, L.; Li, J. Toehold-Initiated Rolling Circle Amplification for Visualizing Individual MicroRNAs In Situ in Single Cells. Angew. Chem. Int. Edit. 2014, 53, 2389-2393. (17) Zhang, Z.; Liu, Y.; Liu, P.; Yang, L.; Jiang, X.; Luo, D.; Yang, D. Non-Invasive Detection of Gastric Cancer Relevant D-Amino Acids with Luminescent DNA/Silver Nanoclusters. Nanoscale 2017, 9, 19367-19373. (18) Li, F.; Dong, Y.; Zhang, Z.; Lv, M.; Wang, Z.; Ruan, X.; Yang, D. A Recyclable Biointerface Based on Cross-Linked Branched DNA Nanostructures for Ultrasensitive Nucleic Acid Detection. Biosens. Bioelectron. 2018, 117, 562-566. (19) Hartman, M. R.; Yang, D.; Tran, T. N. N.; Lee, K.; Kahn, J. S.; Kiatwuthinon, P.; Yancey, K. G.; Trotsenko, O.; Minko, S.; Luo, D. Thermostable Branched DNA Nanostructures as Modular Primers for Polymerase Chain Reaction. Angew. Chem. Int. Edit. 2013, 52, 86998702. (20) Liu, X. P.; Hou, J. L.; Liu, J. H. A Novel Single Nucleotide Polymorphism Detection of a Double-Stranded DNA Target by a Ribonucleotide-Carrying Molecular Beacon and Thermostable RNase HII. Anal. Biochem. 2010, 398, 83-92. (21) Zhou, Q.; Zheng, J.; Qing, Z.; Zheng, M.; Yang, J.; Yang, S.; Ying, L.; Yang, R. Detection of Circulating Tumor DNA in Human Blood via DNA-Mediated Surface-Enhanced Raman Spectroscopy of Single-Walled Carbon Nanotubes. Anal. Chem. 2016, 88, 4759-4765. (22) Rydberg, B.; Game, J. Excision of Misincorporated Ribonucleotides in DNA by RNase H (type 2) and FEN-1 in Cell-Free Extracts. Proc. Natl. Acad. Sci. USA 2002, 99, 1665416659. (23) Itaya, M. Isolation and Characterization of a second RNase-H (RNaes-HII) of Escherichia coli K-12 Encoded by the rnhB Gene. Proc. Natl. Acad. Sci. USA 1990, 87, 8587-8591. (24) Ito, H.; Hasegawa, K.; Nishimaki, T.; Hosomichi, K.; Kimura, S.; Ohba, M.; Yao, H.; Onimaru, M.; Inoue, Haruhiro, I. Silver Nanoscale Hexagonal Column Chips for Detecting Cell Free DNA and Circulating Nucleosomes in Cancer Patients. Sci. Rep. 2015, 5, 10455. (25) Wee, E. J. H.; Wang, Y.; Tsao, S. C. H.; Trau, M. Simple, Sensitive and Accurate Multiplex Detection of Clinically Important Melanoma DNA Mutations in Circulating Tumour DNA with SERS Nanotags. Theranostics 2016, 6, 1506-1513. (26) Ma, W.; Sun, M.; Xu, L.; Wang, L.; Kuang, H.; Xu, C. A SERS Active Gold Nanostar Dimer for Mercury Ion Detection. Chem. Commun. 2013, 49, 4989-4991. (27) Zhuang, H.; Wang, Z.; Zhang, X.; Hutchison, J. A.; Zhu, W.; Yao, Z.; Zhao, Y.; Li, M. A Highly Sensitive SERS-Based Platform for Zn(II) Detection in Cellular Media. Chem. Commun. 2017, 53, 1797-1800. (28) Li, F.; Wang, J.; Lai, Y.; Wu, C.; Sun, S.; He, Y; Ma, H. Ultrasensitive and Selective Detection of Copper (II) and Mercury (II) Ions by Dye-Coded Silver Nanoparticles-Based SERS Probes. Biosens. Bioelectron. 2013, 1, 82-87. (29) Li, A. K.; Tang, L. J.; Song, D.; Song, S. S.; Ma, W.; Xu, L. G.; Kuang, H.; Wu, X. L.; Liu, L. Q.; Chen, X.; Xu, C. A SERS-Active Sensor Based on Heterogeneous Gold Nanostar Core-Silver Nanoparticle Satellite Assemblies for Ultrasensitive Detection of AflatoxinB1. Nanoscale 2016, 8, 1873-1878. (30) Yang, K.; Hu, Y. J.; Dong, N. A Novel Biosensor Based on Competitive SERS Immunoassay and Magnetic Separation for Accurate and Sensitive Detection of


24

Page 25 of 26 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


Chloramphenicol. Biosens. Bioelectron. 2016, 80, 373-378. (31) Ding, S.Y.; You, E. M.; Tian, Z. Q.; Moskovits, M. Electromagnetic Theories of SurfaceEnhanced Raman Spectroscopy. Chem. Soc. Rev. 2017, 46, 4042-4076. (32) Kho, K. W.; Dinish, U. S.; Kumar, A.; Olivo, M. Frequency Shifts in SERS for Biosensing. ACS Nano 2012, 6, 4892-4902. (33) Tang, B.; Wang, J.; Hutchison, J. A.; Ma, L.; Zhang, N.; Guo, H.; Hu, Z.; Li, M.; Zhao, Y. Ultrasensitive, Multiplex Raman Frequency Shift Immunoassay of Liver Cancer Biomarkers in Physiological Media. ACS Nano 2016, 10, 871-879. (34) Guerrini, L.; Pazos, E.; Penas, C.; Vázquez, M. E.; Mascareñas, J. L.; Alvarez-Puebla, R. A. Highly Sensitive SERS Quantification of the Oncogenic Protein c-Jun in Cellular Extracts. J. Am. Chem. Soc. 2013, 135, 10314-10317. (35) Xie, D.; Zhu, W. F.; Cheng, H.; Yao, Z. Y.; Li, M.; Zhao, Y. L. An Antibody-Free Assay for Simultaneous Capture and Detection of Glycoproteins by Surface Enhanced Raman Spectroscopy. Phys. Chem. Chem. Phys. 2018, 20, 8881-8886. (36) Zhu, W.; Cheng, L.; Li, M.; Zuo, D.; Zhang, N.; Zhuang, H.; Xie, D.; Zeng, Q.; Hutchison, J. A.; Zhao, Y. L. Frequency Shift Raman-Based Sensing of Serum MicroRNAs for Early Diagnosis and Discrimination of Primary Liver Cancers. Anal. chem. 2018, 90, 1014410151. (37) Grubisha, D. S.; Lipert, R. J.; Park, H. Y.; Driskell, J.; Porter, M. D. Femtomolar Detection of Prostate-Specific O] An Immunoassay Based on Surface-Enhanced Raman Scattering and Immunogold Labels. Anal. Chem. 2003, 75, 5936-5943. (38) !^ 7, B.; Londergan, C. H.; Webb, L. J.; Cho, M. Vibrational Probes: From Small Molecule Solvatochromism Theory and Experiments to Applications in Complex Systems. Acc. Chem. Res. 2017, 50, 968-976. (39) Capaldo, P.; Alfarano, S. R.; Ianeselli, L.; Zilio, S. D.; Bosco, A.; Parisse, P.; Casalis, L. Circulating Disease Biomarker Detection in Complex Matrices: Real-Time, In Situ Measurements of DNA/miRNA Hybridization via Electrochemical Impedance Spectroscopy. ACS Sens. 2016, 1, 1003-1010. (40) Hodkiewicz, J.; Deck, F. Evaluating Spectral Resolution on a Raman Spectrometer. Thermo Fisher Scientific: Madison, WI, 2010. (41) Lessard-Viger, M.; Rioux, M.; Rainville, L.; Boudreau, D. FRET Enhancement in Multilayer Core-Shell Nanoparticles. Nano Letters 2009, 9, 3066-3071. (42) Viger, M. L.; Brouard, D.; Boudreau, D. Plasmon-Enhanced Resonance Energy Transfer from a Conjugated Polymer to Fluorescent Multilayer Core-Shell Nanoparticles: A Photophysical Study. J. Phys. Chem. C 2011, 115, 2974-2981. (43) Liu, M. Z.; Liu, G. Y. Hybridization with Nanostructures of Single-Stranded DNA. Langmuir 2005, 21, 1972-1978.


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Ultrasensitive Detection of Circulating Tumor DNA of Lung Cancer via

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