Ultrasensitive Detection of Serum MicroRNA Using Branched-DNA

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Biological and Medical Applications of Materials and Interfaces

Ultrasensitive Detection of Serum MicroRNA Using BranchedDNA based SERS Platform Combining Simultaneous Detection of #-Fetoprotein for Early Diagnosis of Liver Cancer Linxiu Cheng, Zhikun Zhang, Duo Zuo, Wenfeng Zhu, Jie Zhang, Qingdao Zeng, Dayong Yang, Min Li, and Yuliang Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10252 • Publication Date (Web): 21 Sep 2018 Downloaded from http://pubs.acs.org on September 22, 2018

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ACS Applied Materials & Interfaces

Ultrasensitive Detection of Serum MicroRNA Using Branched-DNA based SERS Platform Combining Simultaneous Detection of α-Fetoprotein for Early Diagnosis of Liver Cancer Linxiu Cheng,†,‡, Δ ,ǀ, Zhikun Zhang, ‖ ,ǀ Duo Zuo, ∏ ,ǀ Wenfeng Zhu, Qingdao Zeng,*,‡ Dayong Yang,*,‖ Min Li,*, † Yuliang Zhao,†,‡

†

Jie Zhang,

†,ǁ

† CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, 19B, Yuquan Road, Shijingshan District, Beijing 100049, China ‡ CAS Key Laboratory of Standardization and Measurement for Nanotechnology, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology (NCNST), Beijing 100190, China ǁ School of Chemical Engineering and Technology, Key Laboratory of Systems Bioengineering (Ministry of Education), Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300072, China ∆ University of Chinese Academy of Sciences, Beijing 100049, China ∏ Department of Clinical Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, Tianjin’s Clinical Research Center for Cancer, Tianjin 300060, China ǀ These authors contributed to this work equally. Corresponding Author: *E-mail: [email protected]; [email protected]; [email protected]

ABSTRACT: We provided an ultrasensitive sensing strategy for microRNA detection by firstly employing branched DNA. With the aid of micro-contact printing, we realized the multiplex sensing of different kinds of liver cancer biomarkers: microRNA and protein, simultaneously. Delicately designed branched-DNA included multiple complementary sticky-ends as probe to microRNA capture and the 1

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double-stranded rigid branched core to increase active sticky-ends distance and expose more DNA probes for sensitivity. The branched-DNA enables 2 orders of magnitude increase in sensitivity for microRNA detection over single-stranded DNA. The limit of detection (LOD) reaches as low as 10 attomolar (S/N=3) for miR-223 and 10-12 M for AFP, respectively. In addition, this system shows high selectivity and appropriate reproducibility [the relative standard deviation (RSD) is less than 20%] in physiological media. Serum samples are tested and the results are in good agreement with current gold-standard methods, electro-chemiluminescence (ECLIA). The results suggest the reliability of this approach in physiological media and show high potential in the sensing of low abundant microRNA in serum, especially for early diagnosis of primary liver cancers.

KEYWORDS: frequency-shift biosensing, SERS, ultrasensitive detection, branched-DNA, liver cancer biomarker.

INTRODUCTION Liver cancer remains the third deadliest cancer worldwide with high mortality-to-incidence ratio (>0.95).

1, 2

Owing to the late onset primary liver cancer

(PLC), surgical resection or liver transplant are often the only viable curative options and 5 year survival rates are only 7 % for hepatocellular carcinoma (HCC).

3, 4

Early

diagnosis of liver cancer is thus the key to reduce the mortality-to-incidence ratio. The current well-established biomarker for HCC is α-fetoprotein (AFP) which is abundant in fetal serum, while low-expressed in serum of healthy people (age> 1).5-9 However, AFP suffers from both low sensitivity and low selectivity particularly in early stages of the disease.10, 11It has been established that abnormal expression of certain combinations of circulating microRNAs (miRNAs) can be a good indication of early onset PLC. However, it is difficult to identify and quantify miRNAs12 because of the attributes of miRNAs including the small size, low abundance, sequence homology among family members and sensibility to degradation. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is considered 2


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as the golden standard detection method, however it suffers from the lack of house-keeping genes in plasma, making proper normalization and comparison between experiments difficult. Though some interesting detection methods are reported recently13-16, the limit of detection is not low enough especially for low-expressed microRNAs. Therefore, accurate and ultrasensitive detection of miRNA is highly desirable for early diagnosis of liver cancer.17-20 Substantial research efforts have been made to realize delicate detection of these cancer biomarkers recently.21-25 However, directly detecting single biomarker is not sufficient for the diagnosis of HCC and its status. Therefore, the simultaneous detection of miRNAs and AFP may become particularly important for diagnosis and prognosis of HCC.26-30 The extremely high (up to 1011) enhancement of molecular Raman scattering due to coupled surface plasmons at nanoscale junctions between noble metal nanoparticles has made surface-enhanced Raman scattering (SERS)

31-37

an efficient method for

direct detection of miRNA. Due to their highly resolved spectra, SERS-based immunoassays present great potential for increased sensitivity and selectivity, especially for multiplex sensing compared to conventional ELISA or luminescence assays. The major challenge of SERS sensing is to ensure that the target signal can be enhanced at an appropriate level and in a reproducible manner. In the typical ‘sandwich’ assay a Raman reporter molecule (a species with high Raman cross-section) binds with the miRNA such the pair reside in a hotspot between two nanoparticles.38 Recently frequency-shift SERS method has been applied to the detection of proteins and peptides, in which the vibrational normal modes of a Raman reporter is modified upon combining with a target biomarker.39-41 The method is constituted of one single reaction step compared to the traditional ‘sandwich’ SERS assays and facilitates quantification of a shift in an intense scattering band, rather than picking out a signal above a baseline. Synthetic DNA nanostructures were highly attractive due to its unique molecular controllability, versatility and programmability.42, 43 In particular, branched DNA has been demonstrated as a versatile building-block to construct DNA materials for a 3



variety of applications such as biosensing and bioimaging..44,

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45

In our research,

branched DNA was comprised of single-strand DNA (ssDNA) sequence as the active sticky-ends and three branches (Y-DNA) with double-strand DNA (dsDNA) core as scaffold. The structure of branched DNA was potentially beneficial to the stability of detection system based on a robust framework with angular flexibility46. Three active sticky-ends could be designed to play different roles: one was used to link with Raman reporters, and the other two could simultaneously recognize and capture miRNA through Watson-Crick base-paring. Moreover, the double-stranded rigid branched core of the branched DNA could increase active sticky-ends distance to expose more DNA probes in detection system for sensitivity according to minimizing lateral interactions47. In this work, the Y-shape branched DNA (Y-DNA) was firstly introduced for miRNA recognizing and capturing with detection limit as low as 10-17 M. To achieve the simultaneous detection of liver cancer biomarkers AFP and miR-223, microcontact printing technique was employed to construct ordered multi-functional domains of different Raman reporters on a dense silver nanoparticle film for purpose of multiplex sensing. Remarkably, the assays in both fetal bovine serum and patient serum demonstrate the potential application of this sensing platform in clinical settings.

EXPERIMENTAL SECTION A full detail of the employed materials and methods is involved in the Supporting Information. SERS substrates used in this work were Silver nanoparticle films (AgNFs) which were prepared according to our previously reported work 40. Different domains of Raman reporters were fabricated on the AgNFs by using microcontact printing technique. Y- DNA/ssDNA or anti-AFP was covalently bound to relative reporter domains on AgNFs for miRNAs and AFP capture, respectively. SERS spectra were collected on a DXR Smart Raman spectrometer. Sample substrates were remained in buffer solution during SERS measurements. For the peak position definition, it should be noted that the peak position gave similar results after extracted either by fitting to b-spline, or to voigt profiles. The Si (111) wafer was used to 4


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calibrate the Raman peak after each sample was measured.

RESULTS AND DISCUSSION Table 1 Sequences of the oligonucleotides used in this investigation. Name

Sequence

Use

ssDNA1

5’-GAAGCTGCCAGTCCAATCCTGTCGCAC-NH2-3’ 5’-GGAGACTAGATCATGTACTGGCAGCTTCTGGG

ssDNA2 ssDNA3 ssDNA4

Oligonucleotides for synthesis of Y-DNA1.

GTATTTGACAAACTGACA-3’ 5’-GTGCGACAGGATTGATGATCTAGTCTCCTGGGG TATTTGACAAACTGACA-3’ 5’-GAAGCTGCCAGTCCAATCCTGTCGCAC-NH2-3’ 5’-GGAGACTAGATCATGTACTGGCAGCTTCAGCCT

ssDNA5

Oligonucleotides for synthesis of Y-DNA2.

ATCCTGGATTACTTGAA-3’ 5’-GTGCGACAGGATTGATGATCTAGTCTCCAGCCT

ssDNA6

ATCCTGGATTACTTGAA-3’

ssDNA-7

5’-TGGGGTATTTGACAAACTGACA-(CH2)6-NH2-3’

For binding with reporter

miR-223

5’-UGUCAGUUUGUCAAAUACCCCA-3’

Target of detection

miR-26a-5p

5’-UUCAAGUAAUCCAGGAUAGGCU-3’

Target of detection

The average SERS enhancement factor for the AgNFs (Figure 1a) used in this work was estimated to be 107.40 Figure 1b shows the SEM image of Si template with 2 µm × 2 µm square patterns. The principle of simultaneous detection of miRNAs and AFP is illustrated in Figure 1c. A Raman tag with high scattering cross-section, p-methoxybenzoic acid (MBA, Figure 1), was first printed onto a dense AgNFs by contacting PDMS stamp coated with MBA on the surface (Chemisorption between MBA and AgNFs occurred by S-Ag bonding). Patterns of 2 µm × 2 µm square domains of MBA could be constructed on the surface in this fashion, as confirmed previously by atomic force microscopy.40 Second Raman tag was linked to the remaining

bare

surface

of

AgNFs

by

simply

dipping

in

5,

5’-dithiobis(succinimidyl-2-nitrobenzoate (DSNB, Figure 1) solutions. By selecting appropriate reaction conditions, Y-DNA or ssDNA, the complementary microRNA strands and anti-AFP for the capture of the targeted biomarkers microRNA and AFP, 5



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were linked to corresponding Raman tags sequentially to obtain the multi-functional domains modified substrate for multiplex detection.

Figure 1 (a) SEM image of the AgNF substrates; (b) Microscope image of the Si template for making PDMS stamp which is used for microcontact printing of Raman reporters on AgNFs. (c) Schematic of AgNF modification with different reporter domains and the subsequent binding with anti-AFP as well as Y-DNA for final assay of both AFP and miRNA.

In initial experiments, unpatterned silver substrates bonded with Y-DNA1 or anti-AFP was firstly utilized to detect miR-223 and AFP in phosphate-buffered saline solutions (PBS) solution, respectively. To obtain the optimal concentration of Y-DNA1 with DSNB/AgNFs for efficient miRNA capture, the Raman spectra of Y-DNA1 6


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modified DSNB/AgNFs were collected upon 10-6 M miR-223 hybridization with a series of concentrations of Y-DNA1 solution (5×10-9 M, 10-8 M, 5×10-8 M, 10-7 M, 5

×10-7 M, 10-6 M, and 2.5×10-6 M). The peak at ca. 1335 cm−1 which corresponded to the symmetric nitro stretch49-51 of DSNB-derived reporter distinctly shifted to lower frequency with increasing Y-DNA1 concentration and then reached a maximum value of |ΔRaman shift| at 5×10-7 M (Figure 2a), then the shift value decreased with increasing Y-DNA1 concentration. This could be due to the smaller space to effectively hybridize with miRNA at higher Y-DNA1 density on surface. 5×10-7 M was thus considered as an optimal concentration for Y-DNA1’s binding to DSNB/AgNFs to reach the highest hybridization efficiency between Y-DNA1 and miR-223. We observed that the peak at ca. 1335 cm−1 before and after hybridization with 10-6 M miR-223 exhibits a downshift of ~2.3 cm-1 (Figure S1a, b). The plot of the absolute value of ∆Raman versus the synthetic miR-223 concentration shows linear semilog relationship over the range of 10-16 M ~10-12 M miR-223 in PBS media. No further shift was observed as the concentration of synthetic miR-223 was higher than 10-12 M, suggesting the saturation at this concentration (Figure 2b). Furthermore, the concentration-dependent experiments of miR-223 solution from 10-18 M to 10-13 M were carried out to determine the limit of detection (LOD) of this sensing. 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. We are not trying to resolve two closely spaced bands, but rather to distinguish two samples with slight differences in the position of the same band like examples involving material stress measurements where bond angles change resulting in the Raman band associated with those bonds moving to slightly different frequency.52 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 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 we used can distinguish peak shifts as small as 0.066 cm-1

53

. In our work, we define the LOD [defined by 3σ/slope55, 56 (σ is the 7



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standard deviation of blank samples, signal to noise ratio: S/N=3)] for the sensor by giving a safe value of 0.3 cm−1 as the minimum measurable shift. The absolute Raman shift was 2.3 cm-1 ± 0.2 cm-1 and 0.5 ± 0.2 cm-1 for 10-13 M and 10-17 M miR-223 solution, respectively (Figure 2c). MiR-223 caused shift at 10−17 M concentration by 0.5 cm−1, and 10−17 M was claimed as a conservative LOD, presenting the ultrasensitive sensing capability by using branched DNA. Similarly, the LOD of Y-DNA2 sensor is 10-17 M (Figure S2a and b).

Figure 2 Semilog plot of peak shifts around 1335 cm−1 for miR-223 in PBS; (a) On unpatterned DSNB/Y-DNA1 substrate with different concentration of the modified Y-type DNA probe (Y-DNA1) ranged from 5×10-9 M to 2.5×10-6 M. (b) On the unpatterned DSNB/Y-DNA1 substrate (range of miR-223 concentration: 10-6 M ~10-16 M). (c) On the unpatterned DSNB/Y-DNA1 substrate (range of miR-223 concentration: 10-13 M ~10-18 M). (d) On the unpatterned DSNB/ssDNA-7 (linear type) substrate (range of miR-223 concentration: 10-10 M ~ 10-16 M). Error bars in (c) and (d) are the standard deviation of six measurements (2 repeated samples at 3 different places on each sample).

8


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As a comparison, linear structure DNA ssDNA-7 which was identical to the single chain of Y-DNA1 (Table 1) but without the branched structures was tested for miR-223 detection. The peak shifted again to lower wavenumber when exposure to miR-223 solution and shows a linear semilog response over the range of 10-16-10-10 M in PBS (Figure 2d). The vibrational frequency of DSNB at the peak of ca. 1335 cm-1 shifted to lower energy by 2.4 ± 0.1 cm−1 and 0.4 ± 0.2 cm−1 when the substrate was exposed to 10-10 M and 10-15 M miR-223 solution, showing the detection limit of 10-15 M considering 3 times of the blank uncertainty 0.1 cm−1. Recognition of target miRNAs to the Y-DNA/ssDNA induced highly reproducible shifts to lower wavenumber in a specific reporter’s bands, constituting the assay. Apparently, the sensor constituted by branched Y-DNA reached lower LOD than ssDNA for microRNA detection. Hybridization of ssDNA with similar length was reported to cause large increases in height (>10 nm), accompanied by expulsion of the water molecules among dsDNA monolayer, with consequent softening the layer.54 The resulting vibrational frequency shifts was attributed to mechanical stretching of the relevant bonds of reporter according to our previous work40. Each Y-DNA-bound reporter possessed two ssDNAs, which was the major difference to ssDNA. It is reasonable that more stretching for the specific reporter’s band was induced when the hybridization of miRNAs to Y-DNA, which resulted in larger Raman shift and thus presented higher sensitivity for the assay. The patterned substrates with DSNB/Y-DNA1 and MBA/anti-AFP bound domains were utilized to simultaneously detect miR-223 and AFP in PBS solution (Figure 3 and Figure S3). Laser excitation over a 10 µm diameter spot allows for simultaneous collecting Raman signals from both domains. Figure 3a and b shows the SERS spectra from the substrate after incubation in PBS solution with concentration of miR-223 ranging from 10-13 M to 10-18 M and AFP from 10−8 to 10−13 M, and a blank control. The MBA peak at ca. 1580 cm-1 corresponding to the symmetric aromatic C−C stretch and the DSNB peak at ca. 1335 cm-1 due to the symmetric nitro stretch are shown in Figure S3, Figure 3a and b, respectively. Raman shifts of same direction (downshift) to the experiments on the individual DSNB (above) and MBA36 reporters 9



Page 10 of 23

confirmed the integrity of the patterned domains on substrate. The absolute value of ∆Raman shifts exhibits again a semilog linear relationship for AFP at 10−8 ~ 10−12 M and for miR-223 at 10-13 M ~ 10-17 M concentration range, respectively, as shown in Figure 3c and d. Regarding the AFP detection, the ca. 1580 cm-1 peak downshifted by 2.1 ± 0.2 cm−1 and 0.4 ± 0.2 cm−1 for the 10-8 M and 10-12 M AFP solution, respectively. While for miR-223 detection, the ca. 1335 cm-1 peak downshifted by 2.2 ± 0.1 cm−1 and 0.4 ± 0.1 cm−1 for the 10-12 M and 10-17 M solution, respectively. These values were similar to the results for individual DSNB reporter as shown in Figure 2c, indicating multiplex detection did not reduce the sensitivity of this assay.

Figure 3 (a) Raman spectra at the spectral region around 1580 cm−1 shows the responses of the AFP to anti-AFP. (b) Raman spectra at the spectral region around 1335 cm−1 shows the responses of the DSNB/Y-DNA1 to miR-223. Semilog plot of peak shift as a function of AFP and miR-223 concentration in PBS on patterned DSNB/Y-DNA1 and MBA/anti-AFP bound substrate for the peak around 1580 cm− 1 (c) and 1335 cm−1 (d), respectively. Error bars are the standard deviation of six measurements (2 repeated samples at 3 different places on each sample).

10


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To test the selectivity of our approach for miR-223 and AFP binding, the PBS solution was spiked with 0.1 mg mL-1 BSA, and the Raman spectra were recorded after patterned substrate with DSNB/Y-DNA1 and MBA/anti-AFP binding domains were exposed to solutions containing miR-223 and BSA (Figure 4a). Expectedly, the DSNB peak shift at ca. 1335 cm-1 for 10-17 M miR-223 solution was similar to the case without BSA (Figure 4b); meanwhile the MBA peak shift shows no response of the BSA to anti-AFP (Figure 4c). While the substrate with DSNB/Y-DNA1 and MBA/anti-AFP bound regions was exposed to the mixture PBS solution containing AFP (10-12 M) and miR-26a-5p (10-7 M), the spectral region around 1335 cm−1 displays no response of the miR-26a-5p to Y-DNA1 (Figure 4e), and Raman shift of the MBA peak for AFP solution was similar to the case without miR-26a-5p (Figure 4f), suggesting a high specificity of this sensing platform.

11



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Figure 4 (a) Overall responses of the patterned substrate with DSNB/Y-DNA1 and MBA/anti-AFP bound regions to the mixture phosphate-buffered saline solutions (PBS) containing miR-223 (10-17 M) and BSA (0.1 mg/mL). (b) Zoom-in of (a) at the spectral region around 1335 cm−1 in response to miRNA-223. (c) Zoom-in of (a) at the spectral region around 1580 cm−1 shows no response of the BSA to anti-AFP. (d) Overall responses of the patterned substrate with DSNB/Y-DNA1 and MBA/anti-AFP bound regions to the mixture phosphate-buffered saline solutions (PBS) containing AFP (10-12 M) and miR-26a-5p (10-7 M). (e) Zoom-in of (d) at the spectral region around 1335 cm−1 displays no response of the miR-26a-5p to Y-DNA1. (f) Zoom-in of (d) at the spectral region around 1580 cm−1 shows the responses of the AFP to anti-AFP.

12


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To access the performance of this assay in physiologically relevant media, we conducted the sensing experiments using the patterned substrate in fetal bovine serum solutions containing AFP (0, 10−8, 10−9, 10−10, 10−11 10−12, and 10−13 M) and miR-223 (0, 10−13, 10−14, 10−15, 10−16, 10−17 and 10−18 M). As shown in Figure 5a and b, the nearly same downshifts compared with that in PBS (Figure 3c and d) for both Y-DNA1/DSNB and anti-AFP/MBA-derived peaks were observed, with subtle downshift for miR-223 at concentration of 10−13 M. It is safe to conclude that this assay possesses same LOD in physiologically relevant media and the RSD is less than 20%.

Figure 5 Semilog plot of peak shift as a function of AFP and miR-223 concentration on patterned DSNB/Y-DNA1 and MBA/anti-AFP bound substrate in model serum FBS for the peak around 1580 cm− 1 (a) and 1335 cm−1 (b), respectively. (c) Semilog plot of peak shift as a function of miR-223 concentration on patterned DSNB/ssDNA-7 in model serum FBS for the peak around 1335 cm−1. Error bars in (a), (b), and (c) are the standard deviation of six measurements (2 repeated samples at 3 different places on each sample). (d) Down-regulation levels of miR-223. Plot of serum miR-223 in normal volunteers (Normal) and hepatocellular carcinoma (HCC) 13



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patients.

Table 2 simultaneous detection of AFP and miR-223 in human serum. Number

CAFP / M(ECLIA)

CAFP / M (detected)

HCC-1

2.2×10-11

2.7×10-11

7.8×10

HCC-2

5.7×10-8

1.0×10-8

2.0×10

HCC-3

1.6×10-10

2.2×10-10

1.4×10

HCC-4

2.7×10-11

3.0×10

HCC-5

5.0×10-11

4.7×10

Normal-1

-

3.3×10

Normal-2

-

2.5×10

Normal-3

-

0.8×10

Normal-4

-

1.3×10

Normal-5

-

1.3×10

-11 -11 -11 -11 -12 -11 -11

CmiR-223 / M (detected)

2.9×10 8.4×10 9.0×10

-14 -14 -14 -14 -15 -13

3.4×10-14 4.4×10 8.1×10 1.5×10

-13 -13 -13

Importantly, the assay sensitivity of miR-223 was improved by 2 orders with a LOD of 10-17 M compared to ssDNA-7 (LOD of 10-15 M) (Figure 5c). More importantly, as a potential clinical test, it is important to evaluate the assay’s capability in human sera. Finally, trace miR-223 in early stage HCC was simultaneously assayed with the well-established biomarker AFP. MiR-223 is down-regulated in hepatocellular carcinoma (HCC) in comparison to healthy control serum. The concentrations of miR-223 and AFP were extracted from the working plots in Figure 5a and b. The serum concentrations of miR-223 were found to be 10-15 ~10-14 M for early HCC patients and 10-13 M for healthy control (Figure 5d). AFP expression level in the serum was also benchmarked against ECLIA (using a Roche E170 MODULAR Immunoassay Analyzer), which was the most commonly adopted clinically technology. The results show excellent correlation (Table 2), confirming the reliability of our assay in clinical serum samples.

14


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CONCLUSION In summary, we have employed branched DNA for the first time and developed an ultrasensitive sensing platform for microRNA detection with sensitivity as low as 10-17 M. Dedicatedly designed branched DNA complementary with sticky-ends to microRNA capture presents 2 orders higher sensitivity than normal ssDNA, which helps better understand the underlying sensing mechanism. Simultaneous detection of miRNA together with the well-established liver cancer biomarker AFP has been easily achieved with the aid of micro-contact printing techniques for multiplex detection. LOD of this platform is as low as 10 attomolar for miR-223 and 10-12 M for AFP, respectively, in physiological media. Five serum samples with different HCC develop staging were tested and the results indicated the reliability of this method via comparison with conventional ECLIA. In addition, this work will bring the application of branched DNA to a new level of performance that will be of enormous interest in the field of biosensing and have potential future application in clinical diagnosis.

ASSOCIATED CONTENT Supporting Information. The Supporting Information involves Experimental section, responses of the unpatterned DSNB/Y-DNA1 substrate to miR-223 and the responses of the unpatterned DSNB/Y-DNA2 substrate to miR-223 in FBS, Figure S1 and Figure S2.

AUTHOR INFORMATION Corresponding Author * E-mail : [email protected]; * E-mail: [email protected]; * E-mail: [email protected] ORCID 15



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ACKNOWLEDGMENTS This work was supported in part by the National Natural Science Foundation of China (Grant Nos. 21621004, 31671012, 21575101 and 21622404,), the National Basic Research Program of China (nos. 2016YFA0200700, 2015CB932004), and Tianjin Medical University Cancer Institute and Hospital Level Program (Grant No. Y1302). We thank Dr. Huaixin Zhao at Tianjin University for his careful proofreading.

CONFLICT OF INTEREST DISCLOSURE The authors declare no competing financial interest.

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Ultrasensitive Detection of Serum MicroRNA Using Branched-DNA

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