Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2018

Supplementary Information

Ultrasensitive SERS detection of highly homologous miRNAs by generating 3D organic-nanoclusters and a functionalized chip with locked nucleic acid probes

Mengting Zhuab, Zhaomei Sunb, Zhen Zhang*b Shusheng Zhang*b

a Shandong Province Key Laboratory of Life-Organic Analysis, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, China.

b Shandong Province Key Laboratory of Detection Technology for Tumor Markers School of Chemistry and Chemical Engineering, Linyi University, Linyi 276005, China.

* Corresponding Authors. E-mail: [email protected], [email protected]

S1 Experimental section S1.1 Reagents and apparatus Reagents: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (EDC) was procured from Sangon Biotech (Shanghai) Co., Ltd. N-octylether and n-octanoic acid were procured from Tokyo Chemical Industry Development Co., Ltd. (Shanghai).

K2PtCl6 and Indium(III) acetate were procured from Shanghai MACKLIN biochemical Co. Ltd. Tin(II) 2-ethylhexanoate and oleylamine were procured from J&K Scientific Ltd. (Beijing, China). 6-Mercapto-1-hexanol (MCH) and Tris(2- carboxy-ethyl) phosphine (TCEP) were procured from Aladdin Reagent Co. Ltd. (Shanghai). Ethylene glycol (EG) was ordered from Tianjin Bodi Chemical Industry Co., Ltd. (China). miRNeasy Mini Kit was purchased from QIAGEN. Deoxynucleotide solution mixture (dNTPs) and phi29 DNA polymerase were acquired from TaKaRa Biotechnology Co. Ltd (Dalian, China). Agarose was procured from Beijing Solarbio Technology Co., Ltd. (China). The human hepatocellular liver carcinoma cell line HepG2 was obtained from Shanghai Bioleaf Biotechnology Company (Shanghai, China). PC-3 and MCF-7 cells were acquired from KeyGEN biotechnology Company (Nanjing, China). The normal human hepatocytes LO2 We are purchased from Silver Amethyst Biotech. Co. Ltd. (Beijing, China). K562 and CEM cells were obtained from

Shanghai Enzyme-linked Biotechnology Co. Ltd. (Shanghai, China). All other reagents used in this study were of analytical grade and utilized without further purification. DNA sequences were purified by high performance liquid chromatography from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. All miRNAs were diluted by ultrapure water dealed with diethyl pyrocarbonate (DEPC) and used for the experiments. They are shown in Table S1 and S2. All experiments were carried out on a laminar flow bench to ensure a clean environment to control the influence of RNase.

Table S1. DNA Sequence Used in This Work.

Note Sequences

A1 5’- A ACT ATA CA A CCT ACT ACC TCA CCG TC -3’

A2 5’-SH- TTT TT GA CGG TGA GGT AGT AGG T -3’

A3 5’- CG TAG TGA GGT GTA GGT TGT -biotin-3’

A4 5’- TAT ACA ACC TA C ACC TCA CTA C- NH2 -3’

A5 5’- P- TA GGT TGT ATA A CCT ACT ACC TCA CCG TC ACT TTG CTC GCT ACG TAG TGA GGT G -3’

A6 5’- TGA GGT AGT AGG TTG TAT AGT T -3’

A7 5’- A ACT ATA CA A CCT ACT ACC TCA CCG TC -3’ * Blue italic letters delegate the LNA-modified sequences; Red letters delegate the modified sequences on the DNA-chip, compared with sequences on the LNAP-chip.

Table S2. MicroRNA Sequence Used in This Work.

Note Sequences

Let-7a 5’-UGA GGU AGU AGG UUG UAU AGU U-3’

Let-7b 5’-UGA GGU AGU AGG UUG UGU GGU U-3’

Let-7c 5’-UGA GGU AGU AGG UUG UAU GGU U-3’

Let-7d 5’-AGA GGU AGU AGG UUG CAU AGU U-3’

Let-7i 5’-UGA GGU AGU AGU UUG UGC UGU U-3’

Instrument: Raman measurements were implemented on a Renisaw Invia Raman spectrometer (RamLab-010) at an excitation laser (633 nm). A microscope (50 × objective) was applied to focalize the incident excitation laser. The Raman spectrometer was calibrated with WiRE Raman Software Version 2.0 (Renisaw Ltd). Transmission electron microscopy (JEOLJEM-2100) provided transmission electron microscopy (TEM) images. Raman spectra were earned by an internal microscope of Laser micro- Raman spectrometer (Renishaw, UK). X-ray diffraction (XRD) was gauged by a Rigaku X-ray diffractometer. Zeta potential was procured by Nano ZS90 (Malvern, UK). Images of fluorescent cells were recorded by an Eclipse Ti-E inverted microscope (Nikon). The electrochemical impedance spectroscopy (EIS) was measured at room temperature by a CHI-660E electrochemical analyzer.

S1.2 Preparation of Pt/Sn-In2O3

[1] Pt/Sn-In2O3 hybrid materials were synthesized on the basis of the references. First, n- octanoic acid (7.2 mmol), indium (III) acetate (1.216 g), n-octylether (20 mL), oleylamine (20 mmol) and tin (II) 2- ethylhexanoate (0.32 mmol) were evenly mixed at 85 ℃ for 0.5 hour (Sn : In = 1: 9, atomic ratio). Second, the mixed liquor was heated at 150 ℃ for 70 minutes via nitrogen protection and vigorously churn at 290 ℃ for 100 minutes to generate Sn-In2O3 nanoclusters. The nanoclusters were depurated by centrifugation with ethanol. Thirdly, K2PtCl6 (0.21 g) was ultrasonically dispersed in ethylene glycol (200 mL). The pH value was moved to 12 in ethylene glycol dissolvant, on based on NaOH solution (2 mol/L). Sn-In2O3 nanoclusters (0.4 g) were put into the mixture. The above solution was sonicated for 10 minutes and was stirred at 140℃ for

200 minutes. Finally, Pt/Sn-In2O3 nanoclusters were obtained after rinsed by ultrapure water and dried in drying oven.

S1.3 Assembly of streptavidin (SA) functionalized Pt/Sn-In2O3.

The above Pt/Sn-In2O3 (1 mg/mL) were dispersed in thioglycollic acid solution (1 mmol/L), then sonicated for 12 h and washed with ultrapure water to gain the carboxyl nanoclusters. These modified nanoclusters were then incubated with SA in 10 mM PBS containing 10 mmol/L EDC, The mixed solution was stirred for 150 minutes.[1] After centrifugation and cleansing with ultrapure water, Sn-In2O3/Pt/SA nanoclusters were successfully achieved.

S1.4 SERS Measurements The objective suspension was casted on the processed chip, and air-dried at room temperature for SERS analysis. SERS spectra were obtained with an excitation laser (633 nm) and the laser power (10 mW) by Raman spectrometer (Renishaw, U.K.). The time of each SERS spectra was 10 s. Line mapping was used by a streamline Raman mapping system, and SERS maps were garnered via regional integration within the characteristic peaks. Based on different sites of each sample, three spectra were assembled, and averaged to transmit the SERS results. Error bars showed the standard deviation from three replicated experiments.

S1.5 XRD of nanoclusters XRD characterization of nanoclusters were recorded by X-ray diffraction signals for crystal structure analysis. As shown in Fig. S1, the synthesized nanoclusters showed the main characteristic diffraction peak of Sn-In2O3. (211), (222), (400), (411) were on account of 2θ values of 21.32 °, 30.60 °, 34.45 °, 37.51 °, 41.74 °, 45.54 °, 50.96 °,

56.13 ° and 60.55 °. (322), (134), (440), (611) and (622) planes of Sn-In2O3 and a highly crystalline cubic bixbyite structure with In2O3 (ICDD PDF No. 6-416). It could imply that tin appeared as a dopant into the indium oxide lattice, and a solid solution of Sn-

In2O3 was generated instead of a mixture of indium oxide and tin oxide, because of the absence of a peak consistent with the crystalline SnO2 phase. It indicated that the Sn-

In2O3 surface can be effectively dispersed Pt nanoparticles.

Pt/Sn-In2O3

Sn-In2O3 * a Pt Pt*

b

（111） (200) Pt

(222) (400) (440) In2O3 (211) (411) (134) (511) （622） (322)

20 30 40 50 60 2 Theta (degress)

Fig. S1 XRD of Sn-In2O3 and Pt/Sn-In2O3.

S1.6 Preparation of LNAP-chip and DNA-chip.

The Au chips were burnished and sonicated, then rinsed with water and dried under N2. The cleaned Au chip was immersed in A2-solution containing TCEP and incubated for

12 hours. The thiol groups of DNA were stimulated by TCEP to form Au-S bond with the chips, and coupled A2 on the Au chip surface. The chips were thoroughly cleansed with PBS to remove non-specifically bound DNA, after immersed in 1 mM MCH for 1 hour to block the surface. The surface of A2-chip was rinsed with PBS, and then A1 modified with LNAs was hybridized with A2-chip at 37 ℃ for 3 hours. The LNAP- chip was obtained after being rinsed with PBS. For comparison, A7 (DNA sequences) was hybridized with A2-chip at 37 ℃ for 3 hours to obtain the DNA-chip.

The surface of A2-chip was rinsed with PBS, and then A1 (or A7) modified with LNAs was hybridized with A2-chip at 37 ℃ for 3 hours. The LNAP-chip and DNA-chip were obtained after being rinsed with PBS.

S1.7 Preparation of 3D organic-nanoclusters. The LNAP-chip was incubated with a solution containing the specific target at 37 ° C.

After the surface of the modified chip was rinsed with PBS, circle-A4A5 and phi29 DNA polymerase were added for the RCA reaction (Fig. S2) at 37 ℃ for 3 hours. The surface of this chip was then rinsed with PBS, to obtain the RCA-chip. Secondly, the RCA-chip was incubated in the solution of A3-nanoclusters for 3 hours in constant temperature shaker. After being washed with PBS, 3D organic-nanoclusters were successfully achieved.

Fig. S2 The RCA reaction was analyzed by 1.0% agarose gel electrophoresis: A1 (a), A2 (b), A5 (c), circle A4A5 (d), the RCA product (e), and the marker (f).

S2. Optimization of the experimental conditions S2.1 Optimization of the amounts of Phi29 DNA polymerase and pH For obtaining the best biosensing property, a sequence of control experiments were devised to optimize Phi29 DNA polymerase and pH. As shown in Fig. S3-A, the Raman intensities augmented speedily while the amount of polymerase raised from 0.05 to 0.5 UμL-1. But after 0.4 UμL-1, the Raman intensity decreased slightly. Accordingly, 0.4 UμL-1 of Phi29 DNA polymerase was considered to be the optimum. The signals of Raman detection were analyzed under different pH conditions. Fig. S3-B exhibited the impacts of pH on the Raman intensity measured by 5.0×10-14 M let-7a. The Raman intensity (ΔI) attained the maximum at pH 7.4. Thus, 0.01 M PBS at pH 7.4 was selected for subsequent experiments.

S2.2 Optimization of the incubation time and the reaction temperature The incubation time and the reaction temperature are two important factors of enzyme bioactivity and DNA hybridization. In Fig. S3-C, the Raman intensity improved speedily with the increase of incubation time, and acquired a plateau after 240 min. So the reaction time of the experiment was controlled at 240 min. In Fig. S3-D, the Raman intensity (ΔI) changed under different temperature conditions from 35 ℃ to 39 ℃. A maximum was gained at 37 ℃. Thus, 37 ℃ was chosen for the following experiments.

Fig. S3 Effect of the reaction time, on the SERS intensity respond of 5.0×10-14 M let- 7a.

S3. Specificity of the 3DONs-LNAP method The high affinity of LNAs, the high affinity of the complementary DNA or RNA, is one of the most important features of the LNAs. The 3DONs-LNAP method was used to detect the SERS signal of let-7 family members (Table 2). The experiments found that this method had a much higher SERS signal than other detection methods of DNA-chip

(Fig. 2B and 2C), for identifying a single base (5.0*10-15 M, let-7 family members), indicating that the 3DONs-LNAP strategy has high specificity.

S4. Cell culture and miRNAs extraction PC-3, MCF-7, K562, HepG2, CEM and LO2 cells were cultured in RPMI 1640 (penicillin, 100 U/mL; streptomycin, 100 μg/mL) supplemented with 10% fetal bovine serum and maintained at 37 °C in a humidified atmosphere with 5% CO2, according to the protocol of American Type Culture Col-lection. HepG2 cells were collected and centrifuged at 5000 rpm for 7 min in a culture medium, washed once with PBS buffer, and then spun down at 5000 rpm for 7 min. The cell pellets were suspended in 800 mL of lysis solution. Total RNA was extracted from the HepG2 cells by using miRNeasy Mini Kit, in accordance with the manufacturer's procedure. The sample of let-7a from these cells was diluted and then analyzed for the subsequent experiments.

S5. Analysis of endogenous miRNA concentration of cells HepG2 cells were used to extract the total RNA using a RNA kit. By the standard addition method, the recoveries for let-7a in total RNA from HepG2 cells were in the range from 87.3 to 115.3% and the RSD is below 14.3% (Table S4), implying that this method could be potentially applied to analyze endogenous miRNA concentration of cells. Based on the above experiment, it was estimated that the concentration of let-7a in the total RNA (0.01 ug uL-1) extracted from HepG2 cells was (3.25 ± 0.61) ×10-13 M through three parallel experiments.

S6. Analysis of real samples

To appraise its viability of clinic analysis, the 3DON-LNAP tactics were applied to measure let-7a in human real serum samples via standard addition calibration. In virtue of the high specificity and sensitivity of the technique, the serum samples were isolated from solid suspension by super centrifugation, then diluted before measurement, spiked with let-7a at three different concentrations (1.0 ×10-15 M, 5.0 ×10-15 M, and 1.0 ×10-14

M). Through SERS determination, the datum were summed up and listed in Table S5. Each sample was surveyed by three parallel tests, recovery ratio ranged from 89.1 to

106.5%, implying that the 3DON-LNAP method could be potentially applied to analyze miRNAs in complex biological samples.

Table S3. Comparison of different detection methods for the assay of let-7a.

Method Transducer Detection limit Enzyme-free signal amplification via hybridization chain reactions Fluorescent sensing 1 pM [2]

Bifunctional strand displacement amplification-mediated hyperbranched Fluorescence 18 pM [3] rolling circle amplification

DNase-I-Assisted Target Recycling Amplification based on nanoplatform of Fluorescence 3.6 pM [4] polydopamine nanosphere/gold nanocluster

Signal amplification by coupling multiple Fluorescence 15 pM [5] isothermal reactions

Discrimination mode via a single‐base Chemiluminescence 200 aM [6] mutated padlock probe

Nanoflower with advanced oxygen Electrochemistry 1.92 fM [7] reduction reaction rerformance

Programmable strand displacement-based Chemiluminescent magnetic separation 7.6 fM [8] imaging

This work SERS-3DON 6 aM

Table S4. Analysis of let-7a in total RNA of cells by the method. (n =3)

let-7a content let-7a content Samples Recovery (%) RSD (%) in total RNA added detected 1 1.0 ×10-15 M 1.26 ×10-15 M 114.6 ± 0.71% 14.3% 2 5.0 ×10-15 M 3.91 ×10-15 M 87.9 ± 0.56% 10.2% 3 1.0 ×10-14 M 9.13 ×10-15 M 92.3 ± 0.81% 13.6%

Table S5. Detection of let-7a in human serum samples by 3DON-LNAP method. (n =3)

let-7a content let-7a content Samples Recovery (%) RSD (%) added detected 1 1.0 ×10-15 M 8.97 ×10-16 M 92.3 ± 0.55% 8.2% 2 5.0 ×10-15 M 3.60 ×10-15 M 84.3 ± 0.61% 5.6% 3 1.0 ×10-14 M 1.31 ×10-14 M 109.7 ± 0.53% 7.1%

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