Sulfur Regulated Boron Nitride Quantum Dots

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Sulfur Regulated BN QDs Electrochemiluminescence with Amplified Surface Plasmon Coupling Strategy for BRAF gene Detection Yang Liu, Mengke Wang, Yixin Nie, Qian Zhang, and Qiang Ma Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00965 • Publication Date (Web): 19 Apr 2019 Downloaded from http://pubs.acs.org on April 19, 2019

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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.

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Analytical Chemistry

Sulfur Regulated BN QDs Electrochemiluminescence with Amplified Surface Plasmon Coupling Strategy for BRAF gene Detection Yang Liua, Mengke Wanga, Yixin Niea, Qian Zhanga and Qiang Maa* a

Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun, 130012,

China

*Corresponding author Tel.: +86-431-85168352

E-mail address: [email protected]

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

Abstract Because BN QD has a wide-gap (5.0-6.0 eV) than other QDs, the edge configurations, chemical functionalities and heteroatom dopants can decrease and regulated the band gap of BN QDs thereby ameliorating the QDs properties. Now, the precisely control and regulation of BN QD is still at the early stage as a challenging task. Therefore, we used thiourea and L-cysteine as different sulfur precursors to regulate BN QDs optoelectronic properties in this study. It is interesting that two kinds of S-regulated BN QDs present significant different electrochemiluminescence (ECL) properties and electro-optical activity. Furthermore, a ratiometric and enzyme-free ECL sensing mode is constructed with the amplified surface plasmon coupled ECL (SPC-ECL) strategy. The proposed DNA sensor can quantify BRAF gene from 1 pmol/L to 1.5 nmol/L with a limit of detection (LOD) of 0.3 pmol/L. The change of BN QDs ECL signal was easily observed with smartphone camera. This work for the first time provides insights into the role of sulfur regulation in enhancing ECL efficiency and electro-optical activity of BN QD.

Keywords: BN QDs, sulfur regulation, surface plasmon coupling ECL, DNA sensor, BRAF gene


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1. Introduction Electrochemiluminescence (ECL) is a kind of electrochemical redox-induced light emission. In the ECL process, an electron transfer reaction generates an excited state of high-energy species and emits light at electrodes.1-3 The excited state can be produced by the reaction of cation and anion radicals generated from the same luminophor, such as the annihilation mechanism, or from two different precursors (luminophors and co-reactants) by a co-reactant mechanism. Compared to photoluminescence, ECL does not require the use of an external source. The time and position of ECL emitting can be controlled selectively with the change of electrode potential.4-6

Until

now,

the

most

frequently

used

ECL

luminophor

is

ruthenium(II)tris(2,2-bipyridyl) (Ru(bpy)32+) due to its high luminescence efficiency, reversible electrochemical behavior and chemical stability. 7-9 Since the ECL emission of Si QDs was reported in 2002,10 many QDs have been developed with as ECL luminophor.11 As an intrinsic characteristic, ECL can be also used to evaluate the luminescence functions and electro-optical ability of QDs. Because of the low ECL efficiency, high biological toxicity (e.g. CdTe QDs), and blue-green emission (e.g. carbon-based QD), the application of QD-based ECL sensing is limited. Therefore, the development of novel QDs with perfect electrochemical properties is the urgent need in the ECL sensing research. Recently, a new class of special zero-dimensional quantum dots is derived from the atomically-thin two-dimensional sheets (also known as 2D-QDs), such as graphene, transition metal dichalcogenide, graphitic carbon nitride, and hexagonal boron nitride. The distinctive characteristics of 2D-QDs both inherit from 2D-sheets and due to prominent edge and quantum confinement effects. Therefore, the ideal electronic, chemical and catalytic properties embodied fully the power of 2D-QDs in sensing, optoelectronics, catalysis, and energy. Among the 2D-QDs, hexagonal boron nitride QD (BN QDs) has similar structure with graphene, which is also called "white graphene". BN QDs has unique physical properties such as high thermal conductivity, optical and electric properties and chemical stability.12-15 The synthesis methods of



BN QDs are mainly divided into top-down method and bottom-up method. In the top-down method, the synthesis of single-layered BN QDs is mainly fabricated from BN sheets16-17. However, the BN QDs prepared from the top-down method had poor uniformity and poor controllability of doping. So, some research group try to use boric acid as precursors to synthesize a high quantum yield of BN QDs via bottom-up pathway.18-20 Compared with other 2D-QDs, there are much less report in the BN QD-based sensing research. The understanding of BN QDs properties is still fragmentary and largely unexploited. Therefore, we report a new strategy to regulate BN QDs band gap and nanostructure to enable high ECL performance in the biosensing application in this study. We used boric acid and melamine as boron source and nitrogen source respectively, and thiourea and L-cysteine as two different sulfur sources to synthesis two kind of sulfur-regulated BN QDs (S-BN QDs). With sulfur regulated, the ECL efficiency, intensity and stability of the two S-BN QDs are greatly improved. Furthermore, we designed a dual-wavelength SPC-ECL ratiometric sensor with S-BN QDs535 nm as a reference and S-BN QDs620 nm as an analytical tag. The target-catalyzed hairpin assembly (CHA) sensing process was employed to sensitively detect target BRAF gene. As a major member of the RAF-MEK-ERK signal transduction pathway, BRAF gene plays an important role in tumor cell proliferation, differentiation and apoptosis. The mutated BRAF gene remains active, interferes with the normal function of the cell signaling chain, and causes cell abnormalities. 58% of patients with papillary thyroid cancer have mutations in the BRAF gene.21-22 CHA is an enzyme-free amplification strategy based on target recycling.23 The most attractive advantages of CHA were its kinetics-controlled reaction and enzyme-free amplification, which avoid such negative factors as high cost and possible false responses due to the inactivation of the enzyme.24,25 As shown in Scheme 1, the sensing system consisted of two hairpin DNAs (H1 and H2) and one target DNA. The BRAF gene was captured with H1 at first. After the H1 changed to the straight chain structure, the exposed sequence further hybridized with H2. Because the straight


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chain structured H1-H2 duplex is more stable than the H1-BRAF gene hybridization, the BRAF gene was replaced form H1-H2 duplex. In the next cycle, the released target DNA was captured again with H1 hairpin. As a result, a few BRAF gene can cause multiple H1-H2 duplex immobilized on the electrode. When H2 hairpin was labeled with Au NPs, the emission light of S-BN QDs620 nm near the Au NPs undergo resonant interactions. The resulted surface plasmon coupled ECL (SPC-ECL)

26,27

achieve continuous amplification of the QD ECL signal.

2. Experiment Section 2.1 Materials Boric acid (H3BO3), Chloroauric Acid (HAuCl4), melamine (C3H6N6), thiourea (CH4N2S), L-cysteine (C3H7NO2S), sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4) and sodium phosphate (Na3PO4) were purchased from Sino-pharm Co. (Shanghai, China). BRAF DNA and hairpin DNA sequences were purchased from Qingke Co., Ltd. (Tianjin, China). (Table S1) All reagent solutions were prepared using ultrapure water (resistivity as 18 MΩ cm-1 at 25oC). Human serum samples were obtained from the China-Japan union hospital of Jilin university. 2.2 Apparatus TEM images of the nanomaterials were obtained with a Hitachi electron microscope operating at 200 kV acceleration voltages. FT-IR, Photoluminescence (PL), and UV–vis absorption spectra were acquired by Thermo Nicolet 360 FTIR spectrometer, Shimadzu RF-5301 PC spectrofluorophotometer and Hitachi UH5300 UV–vis spectrometer, respectively. The CV and EIS data were acquired with a CHI 660B electrochemical workstation with the glassy carbon electrode (GCE) as the working electrode. In the all electrochemical analysis process, a platinum wire and an Ag/AgCl (saturated KCl) electrode was employed as the counter electrode and the reference electrode, respectively. 2.3 Synthesis of S-BN QDs



S-BN QDsT and S-BN QDsL were synthesized by a microwave-assisted hydrothermal method. First, boric acid (1.6 mmol), melamine (0.27 mmol) and thiourea (0.95 mmol), L-cysteine (0.95 mmol) were dissolved in ultrapure water (10 mL) to synthesize S-BN QDsT and S-BN QDsL, respectively. Under the 1000 Hz reflection frequency of microwave, the S-BN QDsT and S-BN QDsL were obtained within 70 min and 120 oC. To remove excess precusor, the solution was The QDs were centrifuged at 5000 rpm for 5 min and washed two times. Quantum yields (QY) of the S-BN QDsT and S-BN QDsL were measured according to the reported quantum yield of quinine sulfate (0.54) in 0.1 mol/L H2SO4.28,29 The QY was calculated according to the follow equation: Yu=YsIu/Is As/Au nu2/ns2

30

where Y represents QY, I represents the measured integrated PL intensity, A represents the ABS intensity, n is the refractive index of the solvent, u is the S-BN QDs, and s represents the standard sample. The ECL efficiency (ΦECL) of QDs was calculated according to the follow equation: ΦECL=Φ0ECL I/I0 Q0/Q

31

where Φ0ECL is the ECL efficiency of the standard (Ru(bpy)3+, 5%), I and I0 are the integrated ECL intensity of the S-BN QDs and the standard, and Q and Q0 the faradic charges (in coulombs) for the investigated and the standard species, respectively. 2.4 Synthesis of Au NPs and Au NPs-hairpin DNA Au NPs were prepared by means of reduction of HAuCl4·4H2O with sodium citrate. Anfter the 150mL HAuCl4·4H2O (1%) solution was heated to reflux, 7.5mL sodium citrate aqueous solution (1%) was added. When the color of the mixture solution changed from yellow to dark red, the obtained Au NPs solution was cooled and stored at 4 oC for further use. To construct the SPC-ECL probe Au NPs-H2, 700


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μL 10 μM thiol-modified hairpin DNA2 was mixed to 1 mL Au NPs. After incubated for 24 h at 25oC, the Au NPs-H2 conjugates were “aged” in 0.1 mol NaCl solution for one day. Then the solution was centrifuged (12000 rpm and 15 min) to remove excess thiol-DNA. At last, the Au NPs-H2 conjugates were washed two times, re-centrifuged, re-dispersed in 2 mL ultrapure water, and stored at 4 oC. 2.5 ECL detection Prior to ECL measurement, GCE was polished with 1.0 and 0.05 μm α-Al2O3 powder, and then ultrasonically cleaned in ethanol and ultrapure water. The GCE was immersed in (0.5%) PDDA for 5 minutes to obtian a PDDA layer. Then 6 μL L-S-BN QDs, 6 μL 0.2 % chitosan (CS) and 6 μL S-BN QDsT were dropped on PDDA modified GCE respectively. After dried by N2, the S-BN QDsT/CS/S-BN QDsL/PDDA/GCE was blocked with 2 wt % BSA solution. In the next step, 10 μL 1μmol carboxyl-modified H1 solutions was dropped to the GCE and incubated with 37 oC and 2 hours to construct H1/S-BN QDsT/CS/S-BN QDsL/PDDA/GCE electrode. The above electrode was immersed with H2-Au NPs and different concentrations of BRAF DNA for 5 minutes at 95 °C and cooled to room temperature for 1 h. When the hybridization was accomplished, the electrode was thoroughly rinsed and dried for further measurement. The ECL signal was acquired by a Hamamatsu CR125 photomultiplier tube (PMT).The voltage of the PMT was set at 900 V with 535 and 620 nm optical filters. The potential on the working electrode was at 0~-2.0 V. 2.6 Visual detection For visual detection, the 7 cm×5 cm sized FTO glass was employed as working electrode. The excitation potential and co-reactant system were set according to above mentioned ECL detection. In order to get ECL visual images, an iphone X smartphone camera with CMOS sensor chip (3840 x 2160 pixels, pixel size = 1.8 μm) was used without other assistant equipment.

3. Results and discussion



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3.1 . Synthesis and Characterizations of S-BN QDs The morphology of two kind S-BN QDs were characterized by TEM and shown in Fig.1. As seen in the Fig.1, S-BN QDs from thiourea precursor (S-BN QDsT) had an average diameter of 9.8 nm with clearly ordered lattice fringes of 0.76 nm. S-BN QDs from L-cysteine precursor (S-BN QDsL) had an average diameter of 9.2 nm and ordered lattice fringes of 0.72 nm. And Au NPs had a size of 15 nm (Fig.S1). The composition and surface chemistry of prepared S-BN QDsT and S-BN QDsL were characterized by X-ray photoelectron microscopy (XPS) and Fourier-transformed infrared spectroscopy (FT-IR). As is shown in Fig.1(B)-(E) and Fig.1(G)-(J), there were five elemental species in the two S-BN QDs as indicated from the five main peaks at 192.3 eV, 285.9 eV, 399.5 eV, 533.5 eV and 168.5 eV in the spectra corresponding to B, C, N, O and S. It is obvious that the different sulfur precursors introduced different contents of N and S elements in QDs. The different element content resulted in the different regulation effect to BN QDs ECL properties. The FT-IR spectrum confirmed the high occupation of oxygen, nitrogen and sulfur-rich groups in the S-BN QDs (Fig S2). The peak at 3195 cm-1 and 3338 cm-1 were assigned to stretching vibration of O-H and N-H, indicating that there were lots of amino and hydroxyl groups on the surface of BN and S-BN QDs. The peak at 1280 cm-1 and 802 cm-1 were attributed to B-N stretching and B-N bending vibration, respectively.

32

And other characteristic peaks were observed at 1184 cm-1 and 1575

cm-1, which represented to –N-B-O- and -N=N=, respectively. Unlike BN QDs, BN QDs possessed the S-H stretching peak at 2363 cm-1.

33

Thiourea and L-cysteine

worked as sulfur precursor to introduce different function groups on the surface of BN QDs. So, the electro-optical characteristics of S- BN QDs were not like each other. Moreover, compared with BN QDs, the addition of more functional groups via reaction with thiourea or L-cysteine can contribute to more intense electron transition for S-BN QDs. The Zeta potentials of S-BN QDs were shown in Fig S2 (B). Due to the presence of –COOH of S-BN QDsL, the Zeta potential of S-BN QDsL (-18 mV) was quite lower than that of S-BN QDsT (-10.8 mV)


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UV-vis and PL spectrum of S-BN QDs were shown to evaluate the relation between the function group of S-BN QDs and their optical properties. The UV-vis spectra of the S-BN QDs (Fig.S3A) showed the absorption peak centered at 206 nm that can be attributed to n-σ* transitions in C-N bonds. The absorption peak of S-BN QDs at 233 nm was due to π-π* transition of C=C bonds. However, the peak at 233 nm of S-BN QDsL was not obvious. It was due to L-cysteine as sulfur precursor introduced C=O bonds, which weaken the effect of π-π* transition. Compared with BN QDs, the interaction of the major absorbing species on the sp2 platform with the sulfur atom in S-BN QDs caused a shift in absorption spectrum.34 The PL spectrum of S-BN QDs (Fig. S4A, B) was obtained at different excitation wavelength from 350 nm to 500 nm. S-BN QDs showed the excitation-dependent emission with different excitation wavelength. The PL emission of S-BN QDs varied from 400 nm to 600 nm. It is due to the different function groups on the S-BN QDs surface. On the one hand, the surface function groups from different sulfur precursor can cause the distinct emission defect trap states.35-36 The full width at half maximum of emission spectrum for S-BN QDs and BN QDs were 22 nm and 77 nm, respectively. It indicated that the S-BN QDs had more uniform size distribution and reliable defect state-luminescence center. The PL quantum yields (QY) of S-BN QDsT and S-BN QDsL were 8.9% and 2.0%, respectively, which were lower than that of BN QDs (10.31%). It also showed that the addition nonradiative pathways were introduced by sulfur dopants. 3.2 ECL behavior of S-regulated BN QDs Fig 2 showed the ECL spectrum and PL spectrum of both S-BN QDs and BN QDs. The PL emission peak of both S-BN QDs was at 400 nm. The ECL emission peak of S-BN QDsT and S-BN QDsL were at 620 nm and 535 nm, respectively. And the PL emission peak of BN QDs was at 380 nm, while the ECL emission peak was at 575 nm. The PL emission wavelength of S-BN QDs and BN QDs were closed, while their ECL emission wavelengths were quite different. The ECL behavior depended strongly on surface states and functional groups. As is shown in Fig. 2(D), differed with the PL generation process under light excitation, the electron transfer occurs on



the QDs surface in the ECL reaction. The result indicated that the doping of sulfur did not affect the internal structure of the QDs. More sulfur atoms were doped to the surface of QDs. The discriminative ECL emission peak of the two S-BN QDs was attributed to the different contents of N element and S element, which resulted in the varied surface state. The ECL emission process included a hole-electron recombination process occurred on the surface of QDs as annihilation and co-reactant mechanisms. The obvious red shift of ECL emission peak was attributed to the surface defects state of S-BN QDs. In the ECL generation way, an electron transfer process can produce the excited state of QDs. So, more active electron transfer on the QDs surface can provide enough energy, and generate brighter ECL signal. Thus, when the QDs surface state had a narrower band gap than the core, a significant red shift in the ECL peak from the photoluminescence peak can be observed.37 Based on the method of tangent UV -vis spectrum38, the band gap of S-BN QDsT (620 nm emission, orange color) and S-BN QDsL (535 nm emission, green color) are calculated at 4.96 eV and 5.25eV, respectively. The wide band gap of QDs corresponded to the short ECL emission wavelength. In Fig 3(A), bare GCE was detected in PBS containing 0.1 mol/L K2S2O8 and the reduction peak of S2O82- was observed at -0.78 V. The reduction peaks of BN QDs, S-BN QDsT and S-BN QDsL were -0.62V, -0.56V and -0.68V in PBS, respectively. When the BN QDs/GCE, S-BN QDsT/GCE and S-BN QDsL/GCE were measured in PBS containing 0.1 mol/L K2S2O8, the wide reduction peaks shifted to-0.75V, -1.125 V and -0.72 V, respectively. Compared to BN QDs, the peak current of S-BN QDs increased. It indicated that S-BN QDs were reduced more easily than BN QDs and generated more excited state species QDs*. The relation of ECL intensity and exciting potential was shown in Fig 3(B). The excitation potential of BN QDs was -1.85 V. After the sulfur regulation, the excitation potential was reduced to -1.75 V. The electrochemical impedance spectroscopy (EIS) was displayed in Fig 3(C). The electron-transfer resistances of S-BN QDsT and S-BN QDsL were 83 Ω and 145 Ω, respectively. The stability of the QDs (Fig 3D) on GCE was studied by a serious of


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measurements (n=11) in phosphate buffer solution (pH=7.4) with 0.1 mol/L K2S2O8. The relative standard deviation (RSD) of S-BN QDsT and S-BN QDsL and BN QDs was 1.47%, 2.03% and 1.5%, respectively. Because much surface capturing centers of excitons on the S-BN QDs surface were created by sulfur regulation effect, the S-BN QDs can capture more holes from co-reactant and thereby provided higher ECL efficiency. As a result, the ECL intensity of of S-BN QDsT and S-BN QDsL increased 1.67 times and 2.59 times to BN QDs, respectively. The ECL efficiencies of QDs were measured according to the co-reactant ECL state. Compared with the ECL efficiency of BN QDs (ΦECL 1.04%), the ECL efficiency of S-BN QDsT (ΦECL 3.78%) and S-BN QDsL (ΦECL 2.69%) increased largely. (Ru(bpy)32+-TPA, ΦECL 5% as the standard). The ECL mechanisms of S-BN QDs were similar to other QDs, such as CdSe QDs,

39

carbon NCs40, Si NCs10,

GQDs41. The ECL mechanisms of the S-BN QDs were formation of the excited state QDs* with the existence of the K2S2O8 co-reactant. The detailed mechanism for ECL generation can be described with the equations below: S-BN QDs +e- → S-BN QDs.-

(1)

S2O82- + e- → SO42- + SO4.-

(2)

SO4.- →SO42- + h*

(3)

S-BN QDs.-+ SO4.- →S-BN QDs* + SO42-

(4)

S-BN QDs* →BN QDs + hν

(5)

Firstly, S-BN QDs was reduced to the corresponding radical anion(S-BN QDs.-). Meanwhile, K2S2O8 was also reduced to produce a strong oxidizing species (SO4.-), which then react with the negtively charged QDs (S-BN QDs.-) by injecting a hole into the HOMO orbital of S-BN QDs.-, producing an excited state of QDs, (S-BN QDs*) which relaxed to the ground state and light emission. 3.3 The SPC-ECL DNA biosensor applications As shown in Fig 4(A), electrochemical impedance spectroscopy (EIS) provided



the impedance changes on the electrode in the modification process. After S-BN QDsL (535 nm emission, green color)/PDDA layer was modified on the GCE, the electron-transfer resistance increased to be 83 Ω. When 0.2% CS and S-BN QDsT-H1 (620 nm emission, orange color) were modified on the electrode, the impedance value increased to 104 Ω. There was an obvious increase at 314.5 Ω when target DNA and Au NPs-H2 conjugate connected on the electrode. In the BRAF detection, H1 can hybridize with target DNA and open the hairpin loop structure. The process can expose the complementary sequence of H1 and Au NPs-H2, resulting in the Au NPs-H2 connecting with H1 through a branch migration process. Since the H1-H2 conjugate was more stable than the H1-target DNA conjugate, Au NPs-H2 can hybridizes with H1 and release BRAF gene. CV was performed to further investigate the ECL properties of DNA sensor. As shown in Fig 4(B), the oxidization peak current response of dual S-BN QDs-based ratiometric sensing system was lower than L-S-BN QDs/PDDA. When the target DNA and Au NPs-H2 were captured with H1, the current response decreased due to the steric hindrance of DNA. In the ECL curve of dual S-BN QDs-based ratiometric sensing system (Fig 4(C)), the ratio of 620/535 nm ECL intensity was 1.23. Because of the SPC-ECL effect between Au NPs and QDs, the S-BN QDsT ECL signal can be easily amplified. Meanwhile, S-BN QDsL is far from Au NPs and has fixed ECL intensity, which can work as a reference in the ratiometric sensor. As a result, the ratio changed to 1.84 (over 1.5 times) in the presence of 1.5 nmol/L target BRAF. Moreover, as shown in Fig 5, the ECL intensity enhancement was also monitored by the smartphone. It is easy to observe the green ECL color of S-BN QDsL and the complex ECL color of dual S-BN QDs-based ratiometric sensing system. With the concentration of target DNA increasing, the orange ECL color becomes brighter due to the SPC-ECL effect. Fig 6 showed the relationship of between the ECL intensity and the concentrations of BRAF gene from 1 pmol/L to 1.5 nmol/L. The ratios of the ECL signal (I620/I535) were obtained from Fig 6 (B). The linear equation between the concentration of BRAF and the ratio of the ECL intensity was Y (I620/I535) = 0.43 CBRAF + 1.30 with the LOD of


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0.3 pmol/L. The correlation coefficient was 0.97. To evaluate the specificity of the sensor, the response of 0.5 nmol/L target DNA and one-base mismatch DNA were investigated under the same conditions. As shown in Fig 7, compared with the target DNA, the mismatch DNA showed the small change compared to the blank. We also compared some methods for the BRAF gene detection in Table S2. The proposed ECL biosensor also showed good sensitivity. The method was applied to the determination of BRAF gene in human serum samples (shown in Table 1). The range of recoveries was from 93.33% ~ 110.00% and the RSD was less than 4.13%. The results indicated that the sensor can be used as a reliable application for the determining BRAF gene.

4. Conclusion In summary, we synthesized two S-regulated BN QDs and constructed a dual-wavelength SPC-ECL sensor based on the different ECL properties of two S-BN QDs. Due to the flexible regulated band gap of BN QDs, the ECL efficiency and electro-optical activity of S-BN QDs can be improved significantly. Based on the SPC-ECL effect, the ECL intensity of S-BN QDs620 nm enhanced gradually with the increase of target DNA. And a true-color ECL imaging strategy for BRAF detection was developed. This new sensing mode present ideal sensitivity and accuracy. The novel type QD-baed ECL sensor has great potential for future bioanalysis application and diagnostics.

Conflicts of interest There are no conflicts of interest to declare. Supporting Information Sequences of the used oligonucleotides in present work, TEM image of Au NPs, FT-IR spectra and Zeta potentials of QDs, UV-vis absorption spectra of QDs, the PL spectrum of QDs, comparison of different methods for the detection of BRAF gene.



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Acknowledgements The authors gratefully acknowledge financial support from Youth Science Fund of Jilin Province (20140520081JH) and “Thirteenth Five Year” Project of the Science and Technology Research in the Education Department of Jilin Province, China.

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FT-Raman, NMR, UV–Visible), NLO, NBO, HOMO-LUMO, Fukui function and molecular docking study of (E)-1-(5-bromo-2-hydroxybenzylidene)semicarbazide, J. Mol. Struct., 2017, 1141, 284. 39. Li L. L., Liu K. P., Yang G. H., Wang C.M., Zhang J. R., Zhu J. J., Fabrication of Graphene–Quantum

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Scheme 1. Schematic illustration for the ECL DNA sensor



Fig 1.TEM images of (A) S-BN QDsT and (F) S-BN QDsL, (B)-(E) XPS spectrum of S-BN QDsT and (G)-(J) S-BN QDsL.


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Fig 2. ECL spectrum and PL spectrum of (A) S-BN QDsT, (B) S-BN QDsL, (C) BN QDs，(D) A summary of the PL and ECL mechanisms of QDs.



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Fig 3. (A) Cyclic voltammograms of bare GCE, BN QDs, S-BN QDsT/GCE and S-BN QDsL/GCE in PBS containing 0.1 mol/L K2S2O8. Inset: BN QDs, S-BN QDsT/GCE and S-BN QDsL/GCE in PBS. (versus Ag/AgCl reference electrode) (B) ECL behavior of S-BN QDsL and S-BN QDsT (C) EIS spectrum of S-BN QDsL and S-BN

QDsT

in

0.1

mol/L

KCl

solution

containing

2.5

mmol/L

K3[Fe(CN)6]/K4[Fe(CN)6]. (D) The stability of BN QDs, S-BN QDsL and S-BN QDsT.


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Fig 4. (A) EIS spectrum of different electrodes in 0.1 mol/L KCl solution containing 2.5 mmol/L K3[Fe(CN)6]/K4[Fe(CN)6]. The impedance spectra were recorded in the frequency range of 0.01 Hze 100 kHz with the amplitude of 5 mV. Cyclic voltammetry of the DNA biosensor in 0.1 mol/L KCl solution containing 1.0 mmol/L K3[Fe(CN)6]/K4[Fe(CN)6]. (versus Ag/AgCl reference electrode) (C) ECL curves of S-BN QDsT /0.2% CS/ S-BN QDsL (red curve) and 1.5 nmol/L target DNA/Au NPsH2/ S-BN QDsT-H1/0.2% CS/ S-BN QDsL (black curve).



Fig. 5 The digital image of (A) S-BN QDsL, (B) S-BN QDsT-H1/0.2% CS/ S-BN QDsL,(C) 1.0 nmol/L target DNA/Au NPs- H2/ S-BN QDsT-H1/0.2% CS/ S-BN QDsL, (D) 1.5 nmol/L target DNA/Au NPs- H2/ S-BN QDsT-H1/0.2% CS/ S-BN QDsL. ECL curves of (E) S-BN QDsL, (F) S-BN QDsT-H1/0.2% CS/ S-BN QDsL, (G) 1.0 nmol/L target DNA/Au NPs- H2/ S-BN QDsT-H1/0.2% CS/ S-BN QDsL, (H) 1.5 nmol/L target DNA/Au NPs- H2/ S-BN QDsT-H1/0.2% CS/ S-BN QDsT


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Fig 6. (A) ECL traces of target DNA/Au NPs- H2/ S-BN QDsT-H1/0.2% CS/ S-BN QDsT of different concentrations of target DNA. (From left to right: 0.001, 0.01, 0.1, 0.3, 0.5, 0.8, 1.0, 1.5nmol/L. (B) Calibration curve for quantification of target DNA.



Fig 7. Interference of one-base-mismatched DNA with the ECL response pH 7.4 phosphate buffer solution containing 0.1 mol/L K2S2O8.From left to right: blank, 0.5 mol/L one-base-mismatched DNA, 0.5 mol/L target DNA.


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Table 1 Results of determination of target DNA in human serum samples. Sample

Spiked (nmol/L)

Founded (nmol/L) Recovery (%)

RSD (%) (n=3)

1

0.10

0.11

110.00

4.13

2

0.30

0.28

93.33

2.56

3

0.80

0.83

103.75

3.15



For TOC only:


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Sulfur Regulated Boron Nitride Quantum Dots

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