Low-Fouling Surface Plasmon Resonance Sensor for Highly Sensitive

Oct 11, 2018 - The lowestrecorded water level on the Rhine River, caused by a drought in Western Europe, has slowed... POLICY CONCENTRATES ...
0 downloads 0 Views 2MB Size
Download PDF
Subscriber access provided by UNIV OF LOUISIANA

Article

Low-Fouling Surface Plasmon Resonance Sensor for High Sensitive Detection of MicroRNA in Complex Matrix based on DNA Tetrahedron Wenyan Nie, Qing Wang, Liyuan Zou, Yan Zheng, Xiaofeng Liu, Xiaohai Yang, and Kemin Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02686 • Publication Date (Web): 11 Oct 2018 Downloaded from http://pubs.acs.org on October 11, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8 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

Analytical Chemistry

Low-Fouling Surface Plasmon Resonance Sensor for High Sensitive Detection of MicroRNA in Complex Matrix based on DNA Tetrahedron Wenyan Nie, Qing Wang*, Liyuan Zou, Yan Zheng, Xiaofeng Liu, Xiaohai Yang and Kemin Wang* State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, China. ABSTRACT: Antifouling surfaces which could reduce nonspecific adsorption from complex matrix are a great challenge in surface plasmon resonance (SPR) sensors. An antifouling surface made by the covalent attachment of DNA tetrahedron probes (DTPs) on gold surface demonstrated superior antifouling property against protein and cell. DTPs modified Au (DTPs-Au) film for two single protein samples (1 mg/mL myoglobin, 48 mg/mL HSA) and five complex matrices (100% serum, 100% plasma, 9.85×108 red cell numbers/mL, 5% whole blood and cell lysate) had low or ultralow adsorption amounts (≤ 8.0 ng/cm2). More interestingly, DTPs-Au film could also avoid Au deposition on the surface in the process of the catalytic growth of gold nanoparticles (AuNPs). Thus, lowfouling and sensitive SPR sensor for miRNA detection in complex matrix was developed by integrating DTPs-Au film with the catalytic growth of AuNPs. Exploiting the amplification of catalytic growth of AuNPs, the detection limit was 0.8 fM toward target let-7a. Moreover, the SPR sensor revealed excellent selectivity and could distinguish let-7a from homologous family. More importantly, the SPR sensor could be feasible for determining miRNA in 100% human serum and cancer cell lysates, and the results of detecting miRNA from cancer cells were in excellent accord with the ones obtained using qRT-PCR. This assay may provide a great potential as miRNA quantification method in complex samples.

Surface plasmon resonance (SPR) sensor is a powerful technique for real-time monitoring the change of refractive index near the surface.1 Due to its unique property for monitoring molecular interactions, SPR sensor has been widely applied in pharmacy, diagnosis, food science, and environmental monitoring.2-4 However, owing to nonspecific interactions in serum, cell lysate and other biological samples, SPR sensor still suffered from some challenges about biomarkers detection.5-7 For example, since whole blood sample contained abundant proteins and cell matrices, SPR sensor chip could adsorb nonspecifically by these proteins and cell matrices so that it was difficult to distinguish nonspecific signals from those that were analyte triggered.8,9 That is, nonspecific interactions with the SPR chip created false positive responses, hindering the application of SPR sensors for the detection of analytes in crude biological samples. Therefore, a number of researches have pursued the development of antifouling coatings in order to suppress the effect of nonspecific adsorption of protein. Common antifouling coatings in SPR analysis include, but not limited to, alkane thiolate self-assembled monolayers (SAMs),10 zwitterionic compounds,11,12 poly-(ethylene glycol) (PEG) / OEG based materials,13,14 polysaccharide,8,15 amino acids,16 and peptides.17 These rationally designed and synthetic materials possessed satisfactory antifouling performance against protein adsorption. Nonetheless, these antifouling materials suffered from limited stability after functionalization recognition elements, or poor immobilization capacity and poor compatibility to recognition molecules.3,18 For SPR sensing, an ideal antifouling material required excellent antifouling property, high stability, easy process for immobilization, and excellent compatibility to recognition molecules,

as well as low cost and facile fabrication.19 It is still difficult to satisfy all of the above-mentioned requirements. Thus, it was necessary to explore novel antifouling materials. DNA tetrahedron probes (DTPs), due to good mechanical rigidity and structural stability, were widely used in many bioassays, such as electrochemistry sensor,20,21 chemiluminescence biosensor,22,23 fluorescence assay,24,25 dual polarization interferometry,26 SPR biosensor27 and colorimetric method28. Among these assays, DTPs usually played the role of a good sensor substrate, which could improve the efficiency of hybridization as well as lead to a high signal to background ratio.29,30 In addition, few previous works reported that DTPs was highly resistant to protein.31,32 However, DTPs in reducing nonspecific adsorption have been less addressed and needs further exploration in detail. In this study, DTPs modified Au (DTPs-Au) film was used to reduce nonspecific adsorption from complex matrices. More interestingly, it was also found that DTPs-Au film could prevent the deposition of Au in the process of the catalytic growth of gold nanoparticles (AuNPs). As we known, microRNAs are an extensive class of small non-coding RNA that plays a key role in many biological processes, such as cell cycle, apoptosis, organ development, tissue regeneration and aging. Given that the abnormal expression of certain miRNA is closely related to a variety of diseases and disorders, miRNAs become ideal biomarker candidates and therapeutic targets. Herein, microRNA let-7a was chose as a model target, and highly sensitive and low fouling SPR sensor was constructed by integrating DTPs-Au film and the catalytic growth of AuNPs.

ACS Paragon Plus Environment

Analytical Chemistry 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

Page 2 of 8

Figure 1. The schematic illustration of low-fouling SPR biosensor for high sensitive detection of miRNA in complex matrix based on DNA tetrahedron. As shown in Figure 1, DTPs-Au film was first made by modifying DTPs on the Au film and used as capture probes. In the presence of let-7a solution, sandwich structure was formed through DNA hybridization of target let-7a, DNA-linked AuNPs and DTPs-Au film. The SPR signal was enhanced by the electronic coupling between localized plasmon of AuNPs and the surface plasmon wave associated with Au film.33,34 Next, the size of AuNPs was further enlarged by adding the catalytic growth reagents. Once the size of AuNPs was increased, the effect of electronic coupling increased accordingly. The low fouling SPR sensor showed excellent sensitive and selectivity, especially, in real sample detection, suggesting that it has great potential for practical application in clinical diagnosis and biomedical research. EXPERIMENTAL SECTION Self-Assembly of DTPs. DTPs were synthesized by simple denaturing and annealing process.35,36 Equal concentration of four DNA strands (S1, S2, S3, S4, the sequences in the Table S1) in TM buffer (20 mM Tris-HCl, 50 mM MgCl2, pH 8.0) were first mixed with 30 mM TCEP. Here, TCEP was used to cut S-S bond. Next, the mixtures were heated at 95 °C for 5 min and then rapid cooled on ice in 1 min. Once assembled, DTPs consisted of capture DNA at one vertex to anchor the targets and three thiol groups at other three vertices to fixed on Au chip through Au-S bond. Finally, ultrafiltration (50K molecular weight cutoff) was used to purify the obtained DTPs. The synthetic DTPs was characterized by 2% gel electrophoresis in 1× TBE buffer (40 mM Tris-acetate and 1 mM EDTA, pH 8.0) at 90 V for 40 min. The obtained board was visualized under UV irradiation and photographed by using a Molecular Imager Gel Doc XR. Fabrication of SPR Chips. In this work, a surface plasmon resonance spectrometer (EC-SPR1010) (DyneChem, Changchun, China) was used and the angle resolution of EC-SPR1010 system was 0.0015°. EC-SPR 1010 system is a single channel and prism coupling-based instrument equipped with a 650 nm laser as the light source. Au film was first cleaned according to the previous

works.37,38 The Au film was docked into EC-SPR1010 system. Then, 65 μL of the DTPs was dripped to the cleaned Au film surface and allowed to react overnight at room temperature, followed by a thorough rinsing with 10 mM 6 × SSC (0.9 M sodium chloride and 0.09 M trisodium citrate, pH 7.0), and the DTPs-Au chip was obtained. The ssDNA/MCH modified Au (ssDNA/MCH-Au) film was used as contrast. ssDNA was firstly incubated with the clean Au film for 3 h at room temperature. Then, MCH was incubated with ssDNA-modified Au film for 30 min. At last, the ssDNA/MCH-Au film could be obtained by washing with 10 mM 6 × SSC repeatedly. Density Measurement of DTPs on Au Surface. Different concentrations (0.5 μM, 1 μM, 5 μM, and 10 μM) of the DTPs were incubated with Au film for 3 h at 4 °C. After washing with 10 mM PB (pH 7.0) repeatedly, the DTPs-Au films with different density were obtained. Then the surface density of DTPs on the Au film was quantitatively measured with chronocoulometry, as previously described.39 Characterization of Water Contact Angle (WCA). WCA of three kinds of SPR chips (bare Au film, ssDNA/MCH-Au film and DTPs-Au film) were recorded with a video-based contact angle measuring system (DSA100, KRUSS, Germany). 2μL of pure water was dropped on the surfaces and the WCA values were measured by averaging at least five data at different locations for each sample. Measurements of Nonspecific Adsorption. SPR was used to monitor the nonspecific adsorption of seven biological samples on bare Au, ssDNA/MCH-Au and DTPs-Au chips, respectively. Seven samples included two protein solutions (1 mg/mL myoglobin, 48 mg/mL HSA) and five natural complex matrices (100% serum, 100% plasma, 9.85×108 red cell numbers/mL, 5% whole blood and cell lysate). Specifically, myoglobin and HSA were dissolved in a 10 mM PBS (pH 7.0) solution to a final concentration of 1 mg/mL and 48 mg/mL, respectively. Sheep red cell and human

ACS Paragon Plus Environment

Page 3 of 8 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

Analytical Chemistry

whole blood were dissolved in a 10 mM PBS (pH 7.0) solution to a final concentration of 9.85×108 cell numbers/mL and 5%, respectively. 100% blood serum and 100% blood plasma were used without dilution. In brief, a baseline signal was first established by applying a 10 mM PBS buffer (pH=7.0) at a flow rate of 50 μL/min over the SPR chip for 30 min. Then the above biological sample was injected into the flow cell and incubated for at least 30 min. After a thorough rinsing with 10 mM PBS solution for 10 min, the change of SPR angle (Δθ) was recoded. AFM and SEM were used to characterize the nonspecific adsorption of 9.85×108 number/mL red cell and 5% whole blood on bare Au, ssDNA/MCH-Au and DTPs-Au chips by using a scanning probe microscope (SP13800N SPA400, Seiko, Japan) and field emission scanning electron microscope (FESEM, JSM-6700F, JEOL, Japan), respectively. MiRNA Analysis by SPR. Under the temperature of 25°C, different concentration of let-7a solution was added to DTP-Au chip for 50 min. Then 10 mM 6 × SSC was used to wash the sensor chip repeatedly, and the change of resonance angle was recorded, i.e. let-7a was detected by direct hybridization method. Next, DNAlinked AuNPs were added for reacting about 30 min. 10 mM 6 × SSC was used to wash the sensor surface repeatedly, and the resonance angle was recorded, i.e. let-7a was detected using AuNPs amplified SPR sensor. Thirdly, catalytic growth reagent consisting of 3.6 × 10-4 M HAuCl4 and 4 × 10-4 M NADH was added for 20 min. 10 mM 6 × SSC was used to wash the sensor surface repeatedly, and the resonance angle was recorded, i.e. let-7a was detected using DTPs-SPR sensor. Finally, the sensor chip was treated using 0.1% SDS/10 mM NaOH for further use. Cell Extractions and qRT-PCR analysis. Three human cancer cell lines, including breast cancer cell lines (MCF-7), lung cancer cell lines (A549) and hepatocellular carcinoma cell lines (SMMC7721) were employed to prepare the biological samples. The total RNA, including miRNA, was extracted from the human cancer cell lines using a Trizol reagent (Sangon Co.Ltd, Shanghai, China). The concentrated and pure RNA was divided into two equal parts for the detection using qRT-PCR (Sangon Co. Ltd, Shanghai, China) and the DTPs-SPR sensor. The detection of target let-7a in cell extractions followed the above procedures. RESULTS and DISCUSSION Characterization and Modification of DTPs. (i) Electrophoresis characterization. The as-prepared DTPs were validated by 2% gel electrophoresis. As shown in Figure 2A, lane 2, lane3, lane 4, lane 5 and lane 6 showed the band of S2, S1S2, S2S2S3, S1S2S3 and S1S2S3S4 (DTPs), respectively. Other samples migrated faster than DTPs which consisted of four DNA strands, since molecular weight of DTPs was higher than that of other samples. The result demonstrated that DTPs was successfully assembled. (ii) SPR characterization. To monitor the grafting process, the surface modification of Au film was characterized using a commercially available EC- SPR1010 system. The resonance angle change (Δθ), which was larger than 0.0015, was acted as a signal for this SPR instrument. As shown in Figure 2B, after incubated with DTPs, SPR spectrum changed from curve a to curve b and an obvious change of resonance angle (0.3912) was observed, suggesting DTPs could be modified on the Au film surface. (iii) WCA characterization. To characterize the hydrophilicity of DTPs-Au film, static water contact angles of different Au substrates (bare Au film, ssDNA-Au film and DTP-Au film) was measured.

Figure 2. (A) Gel electrophoresis image of (lane 1) Marker, (lane 2) S2, (lane 3) S1+S2, (lane 4) S2+S3+S4, (lane 5) S1+S2+S3, (lane 6) DTPs;(B) SPR spectra of bare Au film (black line) and DTPs-Au film (red line); (C) Contact angles images. As shown in Figure 2C, bare Au film had higher contact angle (95.5°±2.4°) (curve a), while ssDNA/MCH-Au surfaces tended to hydrophilic with water contact angle of 36.2° ±3.6° (curve b). In contrast, attachment of DTPs on Au substrates had a lower contact angle of 19.5°±3.4° (curve c) and showed marked hydrophilicity. Given that many antifouling surfaces in the previous works were hydrophilic,8 it implied that the DTPs-Au film had potential as antifouling surface. Investigation of Nonspecific Adsorption on DTPs-Au Chip. As far as we know, SPR instrument was highly sensitive to nonspecific adsorption and allowed actual detection limit of about 1 ng/cm2.8,40 In this study, the change of resonance angle 0.0015°corresponded to 1.25 ng/cm2. In order to study the antifouling performance of DTPs-Au surface, we used the EC- SPR quantitative analysis mode to detect the adsorption of different proteins on the chip surface. Figure 3 showed SPR signal resulting from nonspecific adsorption of seven samples on different Au substrate. For the bare Au film, a largest SPR response was observed in case of two single protein (1 mg/mL myoglobin, 48 mg/mL HSA) and five complex matrices (100% serum, 100% plasma, 9.85×108 red cell numbers/mL, 5% whole blood and cell lysate) (Figure 3A). That is, if only bare Au film was used for SPR chip, nonspecific interaction of the protein or complex matrices with the Au film created obvious false signal, hindering the application of SPR sensor in the complex matrix. For the ssDNA/MCH-Au film, a larger SPR response was observed in case of above samples (Figure 3B). When the bare Au film was modified using ssDNA/MCH, the amount of nonspecific adsorption on the ssDNA/MCH-Au film could be significantly reduced compared to that on the bare Au film. However, for DTPsAu film, there was almost no change in resonance angle in case of above samples (Figure 3C), suggesting that the amount of nonspecific adsorption on the DTPs-Au film was greatly reduced. In general, there is a proportional relationship between the change of SPR angle and the amount of surface adsorption. Figure 3D and Table S2 (shown in Supporting Information) summarized the nonspecific adsorption of two single protein solutions and five complex matrices on different Au surfaces. For bare Au film, the nonspecific adsorptions from the above biological samples were in the range of 220.0 ng/cm2– 438.0 ng/cm2. For ssDNA/MCH-Au film, the nonspecific adsorptions from these samples were in the

ACS Paragon Plus Environment

Analytical Chemistry

B

500

Bare Au film

2 3

440

C

63.5

64.0

64.5

Angle / degree

4 6 5 72 8

460

440 63.0

65.0

63.5

64.0

1 2 3 4 5 6 7 8

63.0 63.2 63.4 63.6 63.8 64.0 64.2 64.4 64.6

Angle / degree

64.5

65.0

Angle / degree

1 mg/mL Myoglobin 48 mg/mL HSA 100% serum 100% plasma 9.85 x108 red cell number/mL 5% whole blood cell lysate

DTPs-Au film

480

440

1 2 3

D

500

460

ssDNA/MCH-Au film

480

4 5 6 7 8

460

63.0

1 Intensity

Intensity

480

500

ssD N B a r e A /M C H D T P s A u fi -A u f - A u f i il m lm lm

A

Intensity

0

100

200

300

400 2

500

£

Adsorption / (ng/cm )

Figure 3. SPR spectroscopy of buffer (1), 1 mg/mL myoglobin (2), 48 mg/mL HSA (3), 100% serum (4), 100% plasma (5), 9.85×108 red cell number/mL (6), 5% whole blood (7) and cell lysate (8) onto bare Au film (A), ssDNA/MCH-Au film (B) and DTPs-Au film (C). (D) The amount of nonspecific adsorption from several solutions (1 mg/mL myoglobin,48 mg/mL HSA, 100% serum, 100% plasma, 9.85×108 red cell number/mL, 5% whole blood and cell lysate) onto bare Au film, ssDNA/MCH-Au film and DTPs-Au film. The error bars represent the standard deviation of the three independent measurements.

Moreover, the effect of different surface densities of the DTPs on the nonspecific absorption was also investigated. Taking 100% serum and 100% plasma as an example, the amount of protein adsorption decreased with the increase of surface density until the surface density of 5.22 × 1012 molecules per cm2 (shown in Figure 4). Therefore, 5.22 × 1012 molecules per cm2 of DTPs was chosen for the following experiments. Nonspecific adsorption of 9.85×108 numbers/mL red cells and 5% whole blood on bare Au, ssDNA/MCH-Au and DTPs-Au film was investigated by using AFM and SEM images, respectively. After incubation in 9.85×108 numbers/mL sheep red cells or 5% whole blood onto the different Au film, obvious differences among the different Au substrate could be observed even with naked eyes. Bare Au film exhibited cloudy surfaces (shown in Figure 5A1 and 5B1, Figure 6A1and 6B1), indicating significant adsorption of cell

or protein. The ssDNA/MCH-Au film also had a bit of adsorption (Figure 5A2 and 5B2, Figure 6A2and 6B2). However, DTPs-Au film exhibited a very clear surface (Figure 5A3 and 5B3, Figure 6A3and 6B3), indicating no or little adsorption. The results of AFM images and SEM images were in agreement with that of SPR measurements, further illustrating the excellent antifouling ability of DTPs-Au film.

200

100% serum 100% plasma

2

range of 41.5 ng/cm2– 150.3 ng/cm2. While it was found that ultralow adsorptions (≤8.0 ng/cm2) were achieved on DTPs-Au surfaces. Besides, the antifouling capacity of the DTPs-Au film for proteins was comparable to or better than that of reported materials (shown in Table S3 of Supporting Information). Probable causes were attributed to the following two points:8,19 (1) Since DTPs-Au film had a lower contact angle, DTPs have a strong hydration capacity. The surface of DTP-Au film could form a hydration layer which could prevent protein adsorption on the DTPs-Au surface. (2) DTPs, which have special molecular conformations, displays great steric hindrance than ssDNA/MCH, so it could block effectively the nonspecific adsorption.

dsorption/ (ng/cm )

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

Page 4 of 8

150 100 50 0 2.03

4.34

5.22

5.86

Surface density/ 1012 /molecules/cm2 Figure 4. Effect of surface density of DTPs on nonspecific adsorption.

ACS Paragon Plus Environment

Page 5 of 8

B

A

DTPs-Au film

Bare Au film 800

Intensity

ntensity

800

600

2

400

61.5

62.0

62.5

63.0

63.5

64.0

Angle / degree

0.15

61.5

D

62.0

62.5

63.0

10

20

63.5

Angle / degree

64.0

0.10 0.08 0.06

0.10 0.05 0.00

1 2 4 Concentration of (0.18 x HAuCl4 / 0.2 x NADH) mM



Figure 5. Phase-contrast images showing 9.85×108 red cell numbers/mL and 5% whole blood adsorption on (A1, B1) bare Au film, (A2, B2) ssDNA/MCH-Au film and (A3, B3) DTPs-Au film, respectively.

1 2

400

C0.20

600

1



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

Analytical Chemistry

0.04 0.02 0.00 0

Time / min

30

Figure 7. The influence of the catalytic growth reagent on (A) bare Au film and (B) DTPs-Au film surfaces, respectively. Au film was treated without (1) and with (2) the catalytic growth reagent of AuNPs. Effect of the concentration (C) and incubation time (D) of HAuCl4 / NADH.

Figure 6. SEM images showing 9.85×108 red cell numbers/mL and 5% whole blood adsorption on (A1, B1) bare Au film, (A2, B2) ssDNA/MCH-Au film and (A3, B3) DTPs-Au film, respectively. The inhibition of Au deposition by DTPs-Au film. The reduction product of AuCl4- by NADH was deposited on AuNPs, this method was regarded as the catalytic growth of AuNPs.33,34 However, the reduction product of AuCl4- by NADH could be deposited on the Au film surface in the process of the catalytic growth of AuNPs, and it resulted in the increase of background. SiO2coated Au film or layer-by-layer (LBL) assembly modified Au film was used to prevent Au deposition in our previous work.33,34 However, for SiO2 coated Au film, it was prepared by vacuum deposition which need time consuming operations and expensive instruments; for LBL assembly modified Au films, the subsequent modification of the recognition element was time consuming. And the half-width of the SPR spectrum became wider due to the modification of polymer. Here, the inhibition of Au deposition by this DTPs-Au film was investigated. As shown in Figure 7, as bare Au film was incubated with the catalytic growth reagent, a large SPR signal was observed, suggesting that the reduction product of AuCl4- by NADH could deposit on the bare Au film and caused obvious false signal (Figure 7A). That is, bare Au film could not be used for the catalytic growth of AuNPs enhanced SPR sensor. While SPR response was hardly changed as DTPs-Au film was incubated with the catalytic growth reagent (Figure 7B). The results indicated that the DTPs-Au film could prevent the deposition of the reduction product of AuCl4- by NADH, implying that DTPs-Au film could be used for the catalytic growth of AuNPs enhanced SPR sensor.

Different experimental parameters, such as the concentration and the incubation time of HAuCl4 / NADH could influence Au deposition. To eliminate the background interference, the effects of these factors were investigated. The effects of the concentration of HAuCl4 / NADH were firstly investigated. As shown in Figure 7C, SPR signal decreased with the decrease of the concentration of HAuCl4 / NADH, and then Δθ decreased to less than 0.0015° at 20 min. Therefore, 3.6 × 10-4 M HAuCl4 and 4 × 10-4 M NADH was selected to use following experiment. In addition, the effect of incubation time of 3.6 × 10-4 M HAuCl4 and 4 × 10-4 M NADH on Au film surface was investigated. As shown in Figure 7D, SPR signal decreased with the decrease of time, and then Δθ decreased to less than 0.0015° at 20 min. Therefore, 20 min was selected as the optimal incubation time of 3.6 × 10-4 M HAuCl4/ 4 × 10-4 M NADH. Analytical Performance of Let-7a Detection. The feasibility of this method was then investigated by SPR using let-7a as the model target. The result showed that let-7a could be detected by the developed SPR sensor (shown in Figure S1 of Supporting Information). In addition, as shown in Figure S2 of Supporting Information, FE-SEM images showed that the size of AuNPs increased from 13 ± 2 nm (Figure S2A) to 30 ± 4 nm (Figure S2B) after adding the catalytic growth agent. Then the sensitivity of DTPs-Au SPR sensor was investigated. As shown in Figure 8A, SPR spectra were obtained over a 0-2 pM let-7a concentration range. With let-7a concentrations rising, resonance angle gradually increased given that the increased quantity of DNA-linked AuNPs may be captured on Au surface. Figure 8B illustrated that the relation between Δθ and the concentration of let7a of three methods (i.e. direct hybridization measurement, AuNPs amplified SPR and catalytic growth of AuNPs enhanced SPR). It was estimated that catalytic growth of AuNPs enhanced SPR sensor allowed as low as 0.8 fM miRNA was detected (curve c), and AuNPs amplified SPR sensor allowed as low as 50 fM miRNA was detected (curve b), respectively. Therefore, SPR signal enhancement by catalytic growth of AuNPs was more significant than that by AuNPs. In addition, we have compared DTPs-SPR sensor with ssDNA/MCH-SPR sensor. As shown in Figure S3 of Supporting

ACS Paragon Plus Environment

Analytical Chemistry

Information, it was also found that DTPs-SPR sensor was considerably more sensitive than ssDNA/MCH-SPR sensors. Moreover, to further evaluate high amplification effect of the DTPs-SPR sensor, its detection limit and linear range were compared to the previous works (shown in Table S4). The results showed that the DTPs-SPR sensor had excellent sensitivity. Next, the selectivity of DTPs-SPR sensor was studied by using let-7a, let-7e, let-7i, miRNA-429 and random miRNA. As shown in Figure 8C, with the concentration rising, SPR signal almost no changed for non-homologous RNA (such as miRNA-429 and random miRNA), and SPR signal slightly changed for homologous RNA (such as let-7e and let-7i). However, SPR signal evidently changed in case of target let-7a. The results suggested the DTPsSPR sensor could distinguish let-7a from homologous family and showed excellent selectivity. Moreover, the compound sample was also detected using DTPs-SPR sensor for the investigation of selectivity. As shown in Figure S4 of Supporting Information, a clear response was observed in case of target let-7a (200 fM) or a mix of three miRNAs. While slightly signal was observed for either let-7e (2 pM) or let-7i (2 pM). The SPR response caused by let-7a was similar to that caused by the mix of three miRNAs, implying that homologous RNA let-7e and let-7i did not interfere the detection of target let-7a. It further clearly showed that distinct sequence specificity was achieved using the DTPs-SPR sensor. For investigating the reproducibility of DTP-Au chip, 0.1% SDS/10 mM NaOH was exposed to DTP-Au chip. As shown in Figure 8D, when the SPR sensor was used to detect 2 pM let-7a, significant signal degradation was observed until 7 cycles and the standard deviation was about 1% (n=7), demonstrating that DTPsAu chip had good reproducibility. A

B

700

0

2 pM

c

0.20

600



Intensity

0.30 0.25

650

550

0.15

0.05

450

0.00

62.5

63.0

63.5

Angle / degree

b

0.10

500

a

64.0

0.0

0.5

1.0

1.5

0.3 0.30 0.25 0.20 0.15 0.10 0.05 0.00

2.0

2.5

Concentration of miRNA / pM

D

C

2 pM let-7a

0.2





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

Page 6 of 8

- 7a 7 i e l e t l e t- e t-7 2 9 A 4 l A- RN R N mi mi o m d n ra

0

Co

nc

1 0.5 0.2

t en

2

ra

t io

p n/

M

0.1

Blank

0.0 0

2

4

6

Cycle number

8

10

Figure 8. (A) SPR spectra of different concentrations of let-7a. (B) The relationship between Δθ and let-7a concentrations by using different sensing strategies. Direct hybridization measurement (a), AuNPs amplified SPR (b) and the catalytic growth of AuNPs enhanced SPR (c). (C) Selectivity investigation of the DTPs-Au SPR sensor. (D) Reproducibility investigation of the DTPs-Au SPR sensor. Application of the Sensor in Real Samples. For investigating the capacity of the DTPs-SPR sensor in real biological analysis, the cell lysates from three human cancer cell lines, including MCF-7, A549 and SMMC-7721, were detected. The relative expression levels which were used to stand for the levels of let-7a, referred to the expression ratio of let-7a in the target cell lines versus that in

Figure 9. (A) Paired comparison of the let-7a levels detected in the total RNA isolated from three types of human cancer cell lines using qRT-PCR (green histogram) or the proposed sensor (red histogram). Relative signal intensity was based on the expression ratio of let-7a in the target cell lines versus that in the A549 cell lines. (B) Detection of let-7a in 100% human serum (green histogram) and buffer (red histogram). the A549 cell lines. As shown in Figure 9A, the cell lysates from MCF-7 cell lines had higher concentration of let-7a than that from A549 cell lines. Compared to A549 cell lines, concentration of let7a obviously decreased in 7721 cell lines. The results clearly demonstrated the varied contents of let-7a in the cells, which were in good agreement with reported literature.41 In addition, to assess the reliability of this DTPs-SPR sensor, qRT-PCR and the DTPsSPR sensor were used specifically to detect the relative expression levels of let-7a in cell lysates. The results of two methods were in good agreement, which demonstrated the accuracy and practical utility of the proposed method. The DTPs-SPR sensor was also employed to detectlet-7a in undiluted human serum. At first, the RNase inhibitor was used to treat undiluted human serum which contains undetectable miRNA content.42 Then, let-7a undiluted serum sample was acquired by mixing known concentrations of let-7a with undiluted serum sample. The undiluted serum samples which had let-7a were detected. As shown in Figure 9B, whether in 100% human serum or in PBS, the SPR signal caused by the target was almost the same. The results demonstrated that an abundance of protein in the serum did not influence the detection performance of the developed sensor. CONCLUSIONS In summary, a novel antifouling surface was fabricated by coating DTPs to Au surface. SPR, AFM and SEM showed low nonspecific adsorption (≤ 8.0 ng/cm2) onto DTPs-Au surfaces. High sensitive and selective SPR sensor for detection of miRNA in complex matrix was employed integrating DTPs-Au with the catalytic growth of AuNPs. The DTPs-SPR sensor showed high sensitivity and as low as 0.8 fM let-7a was detected. The SPR sensor revealed excellent selectivity and could distinguish let-7a from homologous

ACS Paragon Plus Environment

Page 7 of 8 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

Analytical Chemistry

family. More importantly, the SPR sensor could be feasible for detecting miRNA in undiluted human normal serum and cell lysate. These unique characteristics suggested that the assay would be a promising new way for complex bioanalysis.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Materials and reagents, preparation of AuNPs and assistant DNA-linked AuNPs, investigation of nonspecific adsorption on different Au films, summary of nonspecific adsorption on different antifouling surfaces, characterization of the feasibility, detection performance comparison of our strategy with other SPR method, and selectivity investigation of the DTPs-SPR sensor.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]. Tel./Fax: +86-731-8882 1566 E-mail: [email protected]. Tel/Fax: +86-731-8882 1566

ORCID Qing Wang: 0000-0002-7337-0999 Xiaohai Yang: 0000-0001-8122-7140 Kemin Wang: 0000-0001-9390-4938

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21675047, 21375034 and 21735002), and National Science Foundation for Distinguished Young Scholars of Hunan Province (2016JJ1008).

REFERENCES (1) Wu, B.; Jiang, R.; Wang, Q.; Huang, J.; Yang, X.; Wang, K.; Li, W.; Chen, N.; Li, Q. Chem. Commun. 2016, 52(17): 3568-3571. (2) Zeng, S.; Baillargeat, D.; Ho, H. P.; Yong, K. T. Chem Soc Rev. 2014, 43(10): 3426-3452. (3) Vaisocherová-Lísalová, H.; Surman, F.; Víšová, I.; Vala, M.; Špringer, T.; Ermini, M. L.; Šípova, H.; Šedivak, P.; Houska, M.; Riedel, T.; Pop-Georgievski, O.; Brynda, E.; Homola, J. Anal. Chem. 2016, 88(21): 10533-10539. (4) Masson, J. F. ACS Sens. 2017, 2(1): 16-30. (5) Kim, S.; Lee, H. J. Anal. Chem. 2017, 89, 6624-6630. (6) Hinman, S. S.; McKeating, K. S.; Cheng, Q. Anal. Chem. 2018, 90, 19-39. (7) Kim, S.; Park, J. W.; Wark, A. W.; Jhung, S. H.; Lee, H. J. Anal. Chem. 2017, 89, 12562-12568. (8) Liu, X.; Huang, R.; Su, R.; Qi, W.; Wang, L.; He, Z. ACS Appl. Mater. Interfaces 2014, 6(15): 13034-13042. (9) Blaszykowski, C.; Sheikh, S.; Thompson, M. Chem. Soc. Rev. 2012, 41(17): 5599-5612. (10) Riedel, T.; Riedelova-Reicheltova, Z.; Majek, P.; RodriguezEmmenegger, C.; Houska, M.; Dyr, J.E.; Brynda, E. Langmuir 2013, 29: 3388-3397. (11) Chou, Y. N.; Sun, F.; Hung, H. C.; Jain, P.; Sinclair, A.; Zhang, P.; Bai, T.; Chang, Y.; Wen, T. C.; Yu, Q.; Jiang, S. Acta Biomaterialia 2016, 40: 31-37. (12) Hong, D.; Hung, H. C.; Wu, K.; Lin, X.; Sun, F.; Zhang, P.; Liu, S.; Cook, K. E.; Jiang, S. ACS Appl. Mater. Interfaces 2017, 9, 9255-9259. (13) Zhang, P.; Sun, F.; Hung, H. C.; Jain, P.; Leger, K. J.; Jiang,

S. Anal. Chem. 2017, 89(16): 8217-8222. (14) Sheikh, S.; Yang, D. Y.; Blaszykowski, C.; Thompson, M. Chem. Commun. 2012, 48(9): 1305-1307. (15) Li, Z.; Narouz, M. R.; Munro, K.; Hao, B.; Crudden, C. M.; Horton, J. H.; Hao, H. ACS Appl. Mater. Interfaces 2017, 9(45): 39223-39234. (16) Zou, Q.; Kegel, L.L.; Booksh K. S. Anal. Chem. 2015, 87: 2488-2494. (17) Lin, S.; Zhang, B.; Skoumal, M. J.; Ramunno, B.; Li, X.; Wesdemiotis, C.; Liu, L.; Jia, L. Biomacromolecules 2011, 12, 25732582. (18) Lísalová, H.; Brynda, E.; Houska, M.; Vísova, I.; Mrkvova, K.; Song, X. C.; Gedeonova, E.; Surman, F.; Riedel, T.; Pop-Georgievski, O.; Homola, J. Anal. Chem. 2017, 89(6): 3524-3531. (19) Liu, B.; Liu, X.; Shi, S.; Huang, R.; Su, R.; Qi, W.; He, Z. Acta Biomaterialia 2016, 40: 100-118. (20) Lin, M.; Wang, J.; Zhou, G.; Wang, J.; Wu, N.; Lu, J.; Gao, J.; Chen, X.; Shi, J.; Zuo, X.; Fan, C. Angew. Chem. Int. Ed. 2015, 54(7):2151-2155. (21) Lin, M.; Song, P.; Zhou, G.; Zuo, X.; Aldalbahi, A.; Lou, X.; Shi, J.; Fan, C. Nat. Protoc. 2016, 11(7):1244-1263. (22) Li, M. X.; Feng, Q. M.; Zhou, Z.; Zhao, W.; Xu, J. J.; Chen, H. Y. Anal. Chem. 2018, 90, 1340-1347. (23) Feng, Q. M.; Guo, Y. H.; Xu, J. J.; Chen, H. Y. ACS Appl. Mater. Interfaces 2017, 9, 17637-17644. (24) Zhou, X.; Zhao, M.; Duan, X.; Guo, B.; Cheng, W.; Ding, S.; Ju, H. ACS Appl. Mater. Interfaces 2017, 9, 40087-40093. (25) Xu, F.; Dong, H.; Cao, Y.; Lu, H.; Meng, X.; Dai, W.; Zhang, X.; Al-Ghanim, K. A.; Mahboob, S. ACS Appl. Mater. Interfaces 2016, 8(49): 33499-33505. (26) Wang, S.; Lu, S.; Zhao, J.; Huang, J.; Yang, X. ACS Appl. Mater. Interfaces 2017, 9(47): 41568-41576. (27) Wang, S.; Dong, Y.; Liang, X. Biosens. Bioelectron. 2018, 109, 1-7. (28) Yang, X.; Wen, Y.; Wang, L.; Zhou, C.; Li, Q.; Xu, L.; Li, L.; Shi, J.; Lal, R.; Ren, S.; Li, J.; Jia, N.; Liu, G. ACS Appl. Mater. Interfaces 2017, 9(44): 38281-38287. (29) Wang, J.; Leong, M. C.; Leong, E. Z. W.; Kuan, W. S.; Leong, D. T. Anal. Chem. 2017, 89, 6900-6906. (30) Feng, Q. M.; Zhou, Z.; Li, M. X.; Zhao, W.; Xu, J. J.; Chen, H. Y. Biosens. Bioelectron. 2017, 90: 251-257. (31) Pei, H.; Lu, N.; Wen, Y.; Song, S.; Liu, Y.; Yan, H.; Fan, C. Adv. Mater. 2010, 22(42): 4754-4758. (32) Li, C.; Hu, X.; Lu, J.; Mao, X.; Xiang, Y.; Shu, Y.; Li, G. Chem. Sci. 2018, 9, 979-984. (33) Wang, Q.; Yang, X.; Wang, K. Sensors and Actuators B 2007, 123(1): 227-232. (34) Yang, X.; Wang, Q.; Wang, K.; Tan, W.; Li, H. Biosens. Bioelectron. 2007, 22(6): 1106-1110. (35) Ge, Z.; Lin, M.; Wang, P.; Pei, H.; Yan, J.; Shi, J.; Huang, Q.; He, D.; Fan, C.; Zuo, X. Anal. Chem. 2014, 86(4): 2124-2130. (36) Chen, N.; Qin, S.; Yang, X.; Wang, Q.; Huang, J.; Wang, K. ACS Appl. Mater. Interfaces 2016, 8, 26552-26558 (37) Wang, Q.; Li, Q.; Yang, X.; Wang, K.; Du, S. Zhang, H.; Nie, Y. Biosens. Bioelectron. 2016, 77: 1001-1007. (38) Nie, W.; Wang, Q.; Yang, X.; Zhang, H.; Li, Z.; Gao, L.; Zheng, Y.; Liu, X.; Wang, K. Analytica Chimica Acta 2017, 993: 55-62. (39) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70(22): 4670-4677. (40) Ye, H.; Wang, L.; Huang, R.; Su, R.; Liu, B.; Qi, W.; He, Z. ACS Appl. Mater. Interfaces 2015, 7(40):22448-22457. (41) Tang, Y.; He, X.; Zhenxia Zhou, Z.; Tang, J.; Guo, R.; Feng, X. Chem. Commun. 2016, 52, 13905-13908. (42) Liu, R.; Wang, Q.; Li, Q.; Yang, X.; Wang, K.; Nie, W. Biosens. Bioelectron. 2017, 87: 433-438.

ACS Paragon Plus Environment

Analytical Chemistry 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

For TOC only:

ACS Paragon Plus Environment

Page 8 of 8