Light-Driven Nano-Oscillators for Label-Free Single-Molecule

Subscriber access provided by - Access paid by the | UCSB Libraries

Communication

Light-Driven Nano-Oscillators for LabelFree Single-Molecule Monitoring of MicroRNA Zixuan Chen, Yujiao Peng, Yue Cao, Hui Wang, Jian-Rong Zhang, Hong-Yuan Chen, and Jun-Jie Zhu Nano Lett., Just Accepted Manuscript • Publication Date (Web): 21 May 2018 Downloaded from http://pubs.acs.org on May 21, 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 18 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

Nano Letters

Light-Driven Nano-Oscillators for Label-Free Single-Molecule Monitoring of MicroRNA Zixuan Chen†, Yujiao Peng†, Yue Cao, Hui Wang, Jian-Rong Zhang, Hong-Yuan Chen and JunJie Zhu* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, 163 Xianlin Ave, Nanjing 210023, China. Email: [email protected]

TEL: +(86)2589687204

ABSTRACT: Here we present a mapping tool based on individual light-driven nano-oscillators for label-free single-molecule monitoring of microRNA. This design uses microRNA as a singlemolecule damper for nano-oscillators by forming a rigid dual-strand structure in gap between nano-oscillators and the immobilized surface. The ultrasensitive detection is attributed to comparable dimensions of the gap and microRNA. A developed surface plasmon-coupled scattering imaging technology enables us to directly measure the real-time gap distance vibration of multiple nano-oscillators with high accuracy and fast dynamics. High-level and low-level states of the oscillation amplitude indicate melting and hybridization statuses of microRNA. Life times of two states reveal that the hybridization rate of microRNA is determined by the threedimensional diffusion. This imaging technique contributes application potentials in singlemolecule detection and nano-mechanics study.

ACS Paragon Plus Environment

1

Nano Letters 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 18

KEYWORDS: surface plasmon resonance, nano-oscillators, microRNA, single-molecule detection, dark-field microscopy, mechanoresponsive polymer.

Studies of oligonucleotides hybridization kinetics at the single-molecule level are highly significant for molecular biology and genomic diagnostics.1, 2 A strategy for this aim requires unprecedented temporal resolution, sensitivity and selectivity. Common single-molecule detection techniques include fluorescent microscopy,3-5 ultramicroelectrodes,6, 7 optical absorption microscopy,8, 9 and plasmonic enhancement detection.10-12 However, these methodologies are difficult to attain label-free detection of oligonucleotides due to its low refractive, optical absorption and electroactivity. To date, label-free single-molecule detection of oligonucleotides has only been reported by using carbon nanotube field-effect transistors,13, 14 the nanopore15 and the optical microcavity.16 However, all of them are fabricated based on a single transduce, which lose most spatial resolution information. It is highly desirable to achieve such single-molecule detection abilities in mapping mode for high-throughput analysis of oligonucleotides. Plasmonic nanoparticles have been well developed for highly-sensitive biological sensors due to its stable local surface plasmon resonance (LSPR) scattering, which is sensitive to the adjacent refractive index.17-20 Surface plasmon resonance (SPR)-based nano-oscillators have also been proposed to design highly-sensitive detectors for temporal charge-involved chemical reactions.21, 22

The marriage of two classical plasmonic-based technologies provides the potential possibility

to fabricate an ultrasensitive optical detector for oligonucleotides. For example, the gap distance between the oscillator and the immobilized gold surface might be easily affected by the binding or conformation change of similar sized molecules. The small gap distance change could be


2

Page 3 of 18

accurately evaluated by the LSPR spectral shift of nano-oscillators induced by the coupling effect between plasmonic nanoparticles and the gold surface.23 The issue is that the size of oligonucleotides is usually less than 10 nm,24, 25 and it is still difficult to fabricate and quantitatively measure a sub-nanometer oscillating gap distance to fit this dimension. heating laser

Fcold

G

G

b

c

Gap distance

Fhot

time

Fcold

Fhot

G

G

Fcold

Fhot

G

G

Gap distance

a

time

Gap distance

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

Nano Letters

time pNIPAAm

GNP

DNA

miRNA

Figure 1. Schematic illustration of the single-molecule miRNA detection with light-driven nanooscillators. (a) At low temperature, pNIPAAm molecules have a rigid stretch structure, which supports the gravity of GNPs. Once the light-induced temperature is higher than LCST, pNIPAAm molecules lose the rigid structure, and the gravity makes GNPs fall to the surface. Periodic light illumination induces the oscillation of gap distance. (b) The flexible single-strand DNA has a negligible influence on the oscillation. (c) The hybridization of miRNA forms a rigid duplex structure, which supports the GNP and damps the oscillation. Here we present a light-driven nano-oscillator with a sub-nanoscale gap distance for monitoring single-molecule microRNA (miRNA), which is one of the most important oligonucleotides.2, 26 Each nano-oscillator is comprised with a gold nanoparticle (GNP) and mechanoresponsive


3

Nano Letters 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 18

polymers poly(N-isopropylacrylamide) (pNIPAAm), which have been developed for singlemolecule protein identification.27 The pNIPAAm are self-assembled on the gold-coated glass slide via the Au-S bond, followed by the deposition of GNPs via the Van der Waals' force (Figure 1a). This structure provides a stable environment for GNPs at room temperature (picometer-scale fluctuation). The pNIPAAm molecules act as both the tether and the motor, which have a stretch conformation at room temperature and collapse over the low critical solution temperature (LCST).28 By applying a normal modulated heating laser on the gold surface, pNIPAAm molecules collapse following the periodic photothermal generation, inducing the oscillation of GNPs. For the single-molecule monitoring of miRNA, the complementary strand DNA is immobilized in the gap between GNPs and the gold surface (Figure 1b). The flexible single-strand DNA has a negligible influence on the oscillation. Once miRNA hybridizes to the immobilized DNA, a rigid dual-strand structure is formed, which stops GNPs moving close to the gold surface (Figure 1c). The damping signal in oscillation amplitude time trace enables us to achieve the real-time monitoring of single-molecule hybridization of miRNA. To trace the oscillation amplitude, we develop a surface plasmon-coupled scattering imaging (SPCSI) technology for mapping the LSPR wavelength of GNPs, which has a power law relation with the gap distance.23 As shown in Figure 2a, SPCSI is performed on a home-made coaxial dual-objective microscope. The setup includes an inverted high numerical aperture oil immersion objective below and an upright water immersion objective above. A gold-coated glass slide is placed between two objectives, on which nano-oscillators are self-assembled. A planar surface plasmonic wave is generated on the gold surface by directing a collimated 670 nm p-polarized light onto the glass slide with an appropriate incident angle. The surface plasmonic wave is scattered by GNPs, and then collected by the water-immersion objective to form scattering


4

Page 5 of 18 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

Nano Letters

images. The reflected light is collected by the oil immersion objective to form SPR images.29, 30 Scattering images and SPR images are recorded by two synchronized cameras to obtain two types of images simultaneously (Figure 2b-c). Every GNP in the scattering image has the corresponding tail pattern in the SPR image. To drive the oscillation, we apply a 5 Hz 785-nm heating laser on the gold surface, and some of GNPs demonstrate a distinct amplitude at 5Hz in the scattering intensity (Figure 2d, labeled by arrows). The ratio of oscillating GNPs shows correlation with the pNIPAAm layer density (Figure S1), and at most 60 % GNPs could be driven. b

a

d

Scattering amplitude 4000 3500 3000

Camera 1

2500

60 ×

2000

Water immersion

1500 1000 500

Scattering

0

Surface plasmon wave Reflected

c

e

SPR amplitude 900 800 700

60 ×

600

Oil immersion

500 400

Camera 2

300 200

p-polarized 670-nm light

100

Figure 2. (a) The optical setup of the surface plasmon-coupled scattering imaging system. (b-c) The scattering (b) and SPR (c) image of individual GNPs. (d-e) Scattering amplitude (d) and SPR amplitude (e) image of GNPs in (b) and (c). Scale bar is 10 m. The scattering amplitude of the GNP reduces with increasing heating laser frequency, and a similar trend is observed in the thermal images (Figure S2). We attribute it to the thermal diffusion on the gold surface, because pNIPAAm has a rapid response (< 4ms) to temperature


5

Nano Letters 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 18

change.28, 31 The incident angle of the 670-nm light is also important, Figure 3a displays the incident angle-dependent scattering and SPR intensity of a single GNP in water. The SPR intensity illustrates a typical dip-angle,29 while the scattering intensity shows a peak at the same position. We compare the oscillation amplitude of the GNP at different incident angles. GNPs illustrate similar amplitudes when the incident angle locates at the lower side or the peak position, but the higher incident angle induces a smaller amplitude (Figure 3b). Surface plasmon wave (Esp) is sensitive to the heat generation, which changes the refractive index and thereby the dip-angle.31 The heat gives positive and negative contribution to the scattering oscillation when the angle locates at lower side and higher side of the peak, respectively. To eliminate the heat contribution, we locate the incident angle exactly at the peak position to make Esp insensitive to refractive index. The scattering intensity of GNPs could be converted to the gap distance, because it is determined by Esp and LSPR scattering cross-section (Csca), which are both dependent on the gap distance. Esp decays exponentially from the gold surface into the solution:32, 33 𝑑

𝐸𝑠𝑝 = 𝐸𝑠𝑝 (0)𝑒 − 𝑙

(1)

where d is the gap distance, Esp(0) is the surface plasmon wave at the gold surface, and l (~200 nm) is the decay constant of the surface plasmon wave. The LSPR scattering wavelength peak of GNPs has a power law relation with the distance: peak = d-m.23 Thus, we can express Csca when

peak is located in the range of 630 to 670 nm where Csca has a linear relation with peak:28 𝐶𝑠𝑐𝑎 = 𝛽𝜆𝑝𝑒𝑎𝑘 = 𝛼𝛽𝑑 −𝑚

(2)

where , and m are constant and the scattering intensity Isca can be calculated by 𝑑

𝐼𝑠𝑐𝑎 = 𝐸𝑠𝑝 𝐶𝑠𝑐𝑎 = 𝛼𝛽𝐸𝑠𝑝 (0)𝑒 − 𝑙 𝑑 −𝑚


(3)

6

Page 7 of 18 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

Nano Letters

To measure d of nano-oscillators, we perform following calibration experiments. Initially, we use pNIPAAm with different molecular weight to investigate the d-dependent peak of single GNPs. By recording scattering spectra of at least 100 GNPs on different pNIPAAm-assembled gold surface, a group of Gaussian-shaped distributions of peak was obtained (Figure S3).34 GNPs directly contacting the gold surface illustrate peak at approximately 690 nm, which is much longer than the original value (~530 nm). Once the self-assembled pNIPAAm layer takes GNPs away from the gold surface, peak gradually shifts back. The more molecular weight the pNIPAAm has, the closer peak shifts to the original value. Then we use the atomic force microscope to measure the exact gap distance made by each type of pNIPAAm (Figure S4). We measure the statistic mean height of the pNIPAAm layer using the gold surface as reference, which represents the gap distance. We plot the mean peak to the gap distance (Figure 3c), and the result obeys a power law (peak = 638.4d-0.077, R = 0.99). When d is longer than 10 nm, peak has little change, and Isca is dominated by Esp.21, 22 In contrast, Esp shows neglectful change when d is in the range of 0 to 10 nm in our system, and we have 𝐼𝑠𝑐𝑎 = 𝐴𝑑 −m

(4)

where A and m are constant. We calibrate eq 4 by plotting Isca of 20 GNPs to d (Figure 3d). The power function fitting curve illustrates that A and m are 4134 and 0.616, respectively. Thus, we are able to evaluate the noise level by plotting the FFT spectrum of the gap distance oscillation in Figure 3e, which shows a peak at 10 Hz. The background noise due to the Brownian motion at 10 Hz is 0.007 nm, indicating a detection accuracy of ~0.02 nm (three times the noise level). To attain a sensitive Isca to d, peak of GNPs should be controlled in the range of 630-670 nm due to the linear range of wavelength-dependent scattering cross-section.28 According to experimental results in Figure 3f, pNIPAAm that makes a gap distance of 1.2 nm is selected.


7

Nano Letters

3750

1

2500

0.5

1250

LSPR wavelength (nm)

650 640 630 620 610 600 590 580

0.8 0.6

1.5 2 2.5 3 Gap distance (nm)

2000 1000 Lower angle Dip-angle

Higher angle

Incident angle

6000

Experimental data Fit curve

5000 4000 3000

3.5

Oscillation FFT Background FFT

0.4 0.2 0

3000

2000 1

e Amplitude (nm)

d

Experimental data Fit curve

4000

0

0

Incident angle (°)

c

5000

Scattering amplitude

Scattering intensity

1.5

0

b

5000


104

2

1

f

1.2 Amplitude (nm)

a

SPR Intensity

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 8 of 18

1.5 2 2.5 3 Gap distance (nm)

3.5

pNIPAAm 1 to 5

1.0 0.8 0.6 0.4 0.2

0 10 20 30 40 50 60 70 80 90 100 Frequency (Hz)

0 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 Gap distance (nm)

Figure 3. (a) Incident angle-dependent scattering intensity and SPR intensity of a single GNP. (b) The statistic scattering amplitude of 10 gold nanorods when the incident angle locates at lower angle, higher angle and the SPR dip-angle, respectively. (c) The mean LSPR wavelength of GNPs red-shifts with the decreasing gap distance. (d) Gap distance-dependent scattering intensity of GNPs. (e) Fourier spectrum of the oscillation amplitude of a single GNP. (f) Scattering oscillation amplitudes of GNPs with a series of gap distance illuminated by a 5 Hz heating laser. Next, we use the light-driven nano-oscillators to detect single-molecule miRNA-21. The heating laser is modulated at 10 Hz. Figure 4a presents the scattering amplitude image of GNPs, and most of them illustrate strong oscillations. After incubated with 1 M miRNA-21 at 23 C for 6 min, some of them are damped (Figure 4b and Movie S1), while others remain oscillating. We attribute it to the hybridization of miRNA-21, which are confirmed by the mismatched miRNA


8

Page 9 of 18 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

Nano Letters

(miRNA-199) experiment (Figure S5) and the gel electrophoresis analysis (Figure S6). The different behavior of GNPs in the same condition might be induced by the nonuniform immobilization of complementary DNA, deviations in molecular conformation or the steric effect. Thus, the coverage of probe DNA are optimized for uniform behavior of GNPs. As shown in Figure S7, we compare the probability of binding events in samples with different probe DNA coverage, which is controlled by adjusting the incubation concentration from 25 to 100 M. More probe DNA coverage gives more probability of binding events. But too dense probe DNA also reduce the amount of nano-oscillators due to the displacement of pNIPAAm. Thus, a balanced DNA coverage is constructed with 50 M incubation concentration, which induces 50 % GNPs to oscillate and 85% of them are damped by binding events. In this condition, ~34 DNA strands are located underneath each GNP (Figure S8). Scattering intensity time traces of the three GNPs show oscillation declines successively after the addition of miRNA-21 (Figure 4c). The asynchronous declines reveal that the hybridization of miRNA-21 at different GNPs are uncorrelated. To efficiently trace the real-time oscillation amplitude, the camera is triggered by a pulse signal with twice frequency of that for the heating laser. As a result, scattering images of GNPs with or without laser heating are alternately recorded. By calculating the scattering intensity difference frame by frame, a scattering amplitude image sequence is generated, which enables us to evaluate the amplitude of multiple nano-oscillators simultaneously according to eq 4. Figure 4d displays oscillation amplitude time traces of the three GNPs, all of them initially remain at ~ 0.7 nm, and then successively fall to ~ 0.2 nm within 6 min (at t = 120, 260 and 330 s). Clear stepwise signals reveal hybridization events of single-molecule miRNA-21. The single-molecule signal of miRNA-21 offers a significant improvement of the detection limit, which is attributed to the comparable dimension


9

Nano Letters

of miRNA-21 and the gap distance. With benefits of mapping mode, we observe a large heterogeneity at neighbouring nano-oscillators even if they are exposed to the same concentration miRNA-21. Such huge heterogeneity reveals the importance of high-throughput analysis, which is commonly neglected in common single-molecule detection approaches. Scattering amplitude 5000

1

4500

2

4000 3500 3000 2500 2000

0.6 0.4 0.2 0

f

0

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0

50

100

150

Time (s)

200

1 0.8 0.6 0.4 0.2 0 84 86 88 90 92 94

50

100

150

Time (s)

200

0.8 0.6 0.4

250

100 150 200 250 300 350 Time (s)

200

400

Count

600

0.8 0.6 0.4 0.2 0 0

15

0

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

tmelt (s) 40 35 30

100

Count

2 1.5 1

140

25

80

20

60

15

40

10

20

5

0

50 100 150 200 250 300 350 Time (s)

0.5

120

200 400 600 800 1000

3

2.5

thyb (s)

h

1.8 1.6 1.4 1.2 1

2

3

20

0

800

1

4

5 0

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0

3.5

10

0.2 0

250

50

Count

30 31 32 33 34 35

0

25

Count

0.8

2

30

1

Count

1

g

1

3

1000

1.2

Amplitude (nm)

0.8 0.6 0.4 0.2 0

Amplitude / nm

Amplitude (nm)

e 1.2

5000

1500

3

3

d

Count

2

c

Oscillation amplitude (nm)

b 1


a

Amplitude (nm)

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 10 of 18

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

thyb (s)

0

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

tmelt (s)

Figure 4. (a) Scattering amplitude images of GNPs before miRNA is added. Scale bar is 10 m. Insets: magnification images of the labelled GNPs. (b) Scattering amplitude images of GNPs incubated with miRNA for 6 min. Scale bar is 10 m. Insets: magnification images of the labelled GNPs. (c) Scattering intensity time traces of the three labelled GNPs. (d) Oscillation amplitude time traces of the three labelled GNPs. (e-f) Oscillation amplitude time trace and amplitude-based histograms of time intervals of a nano-oscillator after exposure to 1 M


10

Page 11 of 18 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

Nano Letters

miRNA-21 at 27 (e) and 30 C (f). (g-h). Life time distribution of low-level states and high-level states of a nano-oscillator at 27 (g) and 30 C (h). The decline of oscillation amplitudes becomes reversible at a relative high temperature. Figure 4e displays the amplitude time trace of a GNP at 27 C. Unlike those at 23 C, the amplitude shows intermittent two-level fluctuation instead (more samples in Figure S9). We attribute it to the hybridization (low level) and melting (high level) states of miRNA-21. The fraction of two states show a strong temperature dependence. The amplitude is mostly in the low-level state once miRNA-21 hybridizes to the complementary DNA at 23 C (Figure 4d). Around 27 C, the oscillation amplitude is similarly occupied by two states (Figure 4e). When the temperature increases to 30 C, the low-level state amplitude only shows sharp peaks, and the amplitude mostly remains in the high-level state (Figure 4f). We notice that the temperature making the two sates similarly occupied is lower than the melting temperature Tm in bulk solution (~ 50 C). One possible reason is the photothermal generation, which induces ~8 C above the environment temperature (Figure S1). Interactions between miRNA-21 and the surface also induce a lower melting temperature.13, 35 We extract life times of high (tmelt) and low (thyb) level states of oscillation amplitude to analyze the hybridization kinetics of miRNA-21. tmelt is correlated to the frequency of hybridization events, while thyb indicates the duration of the melting process. Two life times are both dependent on solution temperature. Figure 4g-h illustrates the distribution of tmelt and thyb at 27 and 30 C, respectively. thyb are commonly shorter than 0.2 s and slightly dependent on temperature, indicating the fast melting process of miRNA-21 (Figure 4g). In


11

Nano Letters

remarkable contrast, tmelt shows a broad distribution in the range of 0 to 5 s at 27 C, and decreases to 0.2 s at 30 C (Figure 4h).

Amplitude (nm)

a 1.2

Add 100 nM miRNA-21

1

Add 1 µM miRNA-21

0.8 0.6 0.4 0.2 0

0

50

100

150

200

250

Time (s)

b

40

80

35

70 60

100 nM

25

Count

Count

30 20

40 30

10

20

5

10 0

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

1.2 1

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

tmelt (s)

tmelt (s)

c

1 µM

50

15

0

Amplitude (nm)

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 12 of 18

Add 1 µM single-basemismatched miRNA

Add 1 µM miRNA-21

0.8 0.6 0.4 0.2 0 0

50

100

150

200

250

300

350

400

450

500

Time (s)

d

Type 1 Half Damped

Type 2 Half Damped

Completely damped

Figure 5. (a) Oscillation amplitude time trace of a nano-oscillator after exposure to 100 nM and 1 M miRNA-21 at 30 C. (b) Life time distribution of high-level states in 100 nM miRNA-21, and 1 M miRNA-21, respectively. (c) Oscillation amplitude time trace of a nano-oscillator after exposure to 1 M single-base-mismatched miRNA and 1 M miRNA-21 respectively at 28 C. (d) Schematic illustration of the mechanism of two types of hybridized duplexes-induced oscillation damping.


12

Page 13 of 18 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

Nano Letters

Furthermore, how life times are associated with solution-based kinetics is determined by investigating the concentration-dependence of tmelt. Figure 5a shows the oscillation amplitude time trace of a nano-oscillator exposed to 100 nM and 1 M miRNA-21 successively, and more hybridization events are observed in the higher concentration solution. We compare the tmelt distribution in two conditions (Figure 5b), and find it has a strong concentration dependence. tmelt mostly centers on 0.5 s in low concentration and decreases to 0.2 s in high concentration. Above results reveal that the frequency of hybridization events are dependent on both temperature and target miRNA concentration. Thus, we believe that the hybridization rate is determined by the three-dimensional diffusion rate. The ultimate sensitivity of this method is investigated by monitoring the hybridization events of a single-base-mismatched miRNA strand of miRNA-21 (see in supporting information), for which we observe only half-height declines in the amplitude compared with miRNA-21 (Figure 5c, marked by dotted circles). We attribute the half-height declines to the partial hybridization of single-base-mismatched miRNA strands, because the mismatched base locates in the middle of the full strand. The partial hybridization might make two types of hybridized duplexes (Figure 5d), which both induce a weaker damping effect. The high sensitivity to single-base-matched miRNA offers the ability for single nucleotide polymorphism detection. In conclusion, we have constructed light-driven nano-oscillators for label-free single-molecule monitoring of miRNA. The design is based on the accurate measurement of sub-nanoscale oscillation amplitude of single nano-oscillators, which is performed on a home-made SPCSI system. The ultrasensitive detection of miRNA is attributed to the similar dimension of the gap distance and miRNA molecules. By using this method, we successfully demonstrate that the hybridization kinetics of single-molecule miRNA-21 is determined by the three-dimensional


13

Nano Letters 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 14 of 18

diffusion rate. Thus, the detection limit could be further improved by the flow cell or higher temperature. The large heterogeneity from neighbouring nano-oscillators reveals the significance of the high-throughput single-molecule detection. We also demonstrate the ability of this technology for single nucleotide polymorphism detection. Thus, we believe that the singlemolecule detection strategy has wide potential applications, such as oligonucleotides screening, high-throughput single-molecule studies, and fabrication of nano-mechanics.

ASSOCIATED CONTENT Supporting Information Following files are available free of charge. Supporting Information (PDF) Movie S1: The amplitude decline of a single nano-oscillator. (AVI)

AUTHOR INFORMATION Corresponding Author *Jun-Jie Zhu: 0000-0002-8201-1285 *E-mail: [email protected]. Author Contributions Z. Chen and J.-J. Zhu conceived the study. Z. Chen, Y. Peng and Y. Cao performed the experiments. H. Wang performed the atomic force microscope experiments. Z. Chen built the


14

Page 15 of 18 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

Nano Letters

optical setup. Z. Chen, Y. Peng, Y. Cao and J.-J. Zhu designed the experiments. J.-R. Zhang and H.-Y. Chen advised the experiments. Z. Chen analyzed the data and wrote the paper. †

These authors contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This research is supported by National Natural Science Foundation of China (Grants Nos. 21335004, 21327902, 21427807 and 21605081), Natural Science Foundation of Jiangsu Province (Grants No BK20160638) and China Postdoctoral Science Foundation (Grants Nos. 2016M590434 and 2017T100345). ABBREVIATIONS LCST, low critical solution temperature; SPCSI, surface plasmon-coupled scattering imaging; miRNA, microRNA; GNP, gold nanoparticle. REFERENCES (1) Bartel, D. P. Cell 2009, 136, 215-233. (2) Harvey, J. D.; Jena, P. V.; Baker, H. A.; Zerze, G. H.; Williams, R. M.; Galassi, T. V.; Roxbury, D.; Mittal, J.; Heller, D. A. Nat. Biomed. Eng. 2017, 1, 0041. (3) Johnson-Buck, A.; Su, X.; Giraldez, M. D.; Zhao, M.; Tewari, M.; Walter, N. G. Nat. Biotechnol. 2015, 33, 730-732. (4) Moerner, W. E.; Orrit, M. Science 1999, 283, 1670-1676. (5) Durisic, N.; Laparra-Cuervo, L.; Sandoval-Álvarez, Á.; Borbely, J. S.; Lakadamyali, M. Nat. Methods 2014, 11, 156.


15

Nano Letters 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 16 of 18

(6) Sekretaryova, A. N.; Vagin, M. Y.; Turner, A. P. F.; Eriksson, M. J. Am. Chem. Soc. 2016, 138, 2504-2507. (7) Dick, J. E.; Renault, C.; Bard, A. J. J. Am. Chem. Soc. 2015, 137, 8376-8379. (8) Gaiduk, A.; Yorulmaz, M.; Ruijgrok, P. V.; Orrit, M. Science 2010, 330, 353-356. (9) Celebrano, M.; Kukura, P.; Renn, A.; Sandoghdar, V. Nat. Photon. 2011, 5, 95. (10) Zijlstra, P.; Paulo, P. M.; Orrit, M. Nat. Nanotechnol. 2012, 7, 379-382. (11) Punj, D.; Mivelle, M.; Moparthi, S. B.; van Zanten, T. S.; Rigneault, H.; van Hulst, N. F.; Garcia-Parajo, M. F.; Wenger, J. Nat Nano 2013, 8, 512-516. (12) Yuan, H.; Khatua, S.; Zijlstra, P.; Yorulmaz, M.; Orrit, M. Angew. Chem. Int. Ed. 2013, 52, 1217-1221. (13) Sorgenfrei, S.; Chiu, C.-y.; Gonzalez Jr, R. L.; Yu, Y.-J.; Kim, P.; Nuckolls, C.; Shepard, K. L. Nat. Nanotechnol. 2011, 6, 126-132. (14) Vernick, S.; Trocchia, S. M.; Warren, S. B.; Young, E. F.; Bouilly, D.; Gonzalez, R. L.; Nuckolls, C.; Shepard, K. L. Nat. Commun. 2017, 8. (15) Cao, C.; Ying, Y.-L.; Hu, Z.-L.; Liao, D.-F.; Tian, H.; Long, Y.-T. Nat. Nanotechnol. 2016, 11, 713-+. (16) Baaske, M. D.; Foreman, M. R.; Vollmer, F. Nat. Nanotechnol. 2014, 9, 933-939. (17) Liu, G. L.; Long, Y. T.; Choi, Y.; Kang, T.; Lee, L. P. Nat. Methods 2007, 4, 1015-1017. (18) Mayer, K. M.; Hafner, J. H. Chem. Rev. 2011, 111, 3828-3857. (19) Beuwer, M. A.; Prins, M. W. J.; Zijlstra, P. Nano Lett. 2015, 15, 3507-3511. (20) Shi, L.; Jing, C.; Ma, W.; Li, D.-W.; Halls, J. E.; Marken, F.; Long, Y. T. Angew. Chem. Int. Ed. 2013, 52, 6011-6014. (21) Fang, Y.; Chen, S.; Wang, W.; Shan, X.; Tao, N. Angew. Chem. Int. Ed. 2015, 54, 25382542. (22) Shan, X.; Fang, Y.; Wang, S.; Guan, Y.; Chen, H.-Y.; Tao, N. Nano Lett. 2014, 14, 41514157. (23) Mock, J. J.; Hill, R. T.; Tsai, Y.-J.; Chilkoti, A.; Smith, D. R. Nano Lett. 2012, 12, 17571764. (24) Sonnichsen, C.; Reinhard, B. M.; Liphardt, J.; Alivisatos, A. P. Nat Biotech 2005, 23, 741745. (25) Smith, B. D.; Liu, J. J. Am. Chem. Soc. 2010, 132, 6300-6301.


16

Page 17 of 18 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

Nano Letters

(26) Mitchell, P. S.; Parkin, R. K.; Kroh, E. M.; Fritz, B. R.; Wyman, S. K.; PogosovaAgadjanyan, E. L.; Peterson, A.; Noteboom, J.; O'Briant, K. C.; Allen, A.; Lin, D. W.; Urban, N.; Drescher, C. W.; Knudsen, B. S.; Stirewalt, D. L.; Gentleman, R.; Vessella, R. L.; Nelson, P. S.; Martin, D. B.; Tewari, M. P. Natl. Acad. Sci. USA 2008, 105, 10513-10518. (27) Kusolkamabot, K.; Sae-ung, P.; Niamnont, N.; Wongravee, K.; Sukwattanasinitt, M.; Hoven, V. P. Langmuir 2013, 29, 12317-12327. (28) Chen, X.; Xia, Q.; Cao, Y.; Min, Q.; Zhang, J.; Chen, Z.; Chen, H.-Y.; Zhu, J.-J. Nat. Commun. 2017, 8, 1498. (29) Shan, X.; Patel, U.; Wang, S.; Iglesias, R.; Tao, N. Science 2010, 327, 1363-1366. (30) Shan, X.; Díez-Pérez, I.; Wang, L.; Wiktor, P.; Gu, Y.; Zhang, L.; Wang, W.; Lu, J.; Wang, S.; Gong, Q. Nat. Nanotechnol. 2012, 7, 668-672. (31) Chen, Z.; Shan, X.; Guan, Y.; Wang, S.; Zhu, J.-J.; Tao, N. ACS Nano 2015, 9, 11574– 11581. (32) Jain, P. K.; Huang, W.; El-Sayed, M. A. Nano Lett. 2007, 7, 2080-2088. (33) Wang, S.; Shan, X.; Patel, U.; Huang, X.; Lu, J.; Li, J.; Tao, N. P. Natl. Acad. Sci. 2010, 107, 16028-16032. (34) Chen, Z.; Li, J.; Chen, X.; Cao, J.; Zhang, J.; Min, Q.; Zhu, J. J. J. Am. Chem. Soc. 2015, 137, 1903-1908. (35) Brewood, G. P.; Rangineni, Y.; Fish, D. J.; Bhandiwad, A. S.; Evans, D. R.; Solanki, R.; Benight, A. S. Nucleic Acids Res. 2008, 36.


17

Nano Letters

Table of Contents graphic: 0.8 DNA

miRNA

Amplitude (nm)

GNP

Hybridization

pNIPAAm

Melting

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 18 of 18

Melting

0.6 0.4 0.2 Hybridization 0

30

31 32 33 34 Time (s)


35

18

Light-Driven Nano-Oscillators for Label-Free Single-Molecule

Recommend Documents