Supporting Informations

Transcription

1 Supporting Informations Design of Molecular Beacon as Signaling Probes for ATP Detection in Cancer Cells Based on Chemiluminescence Resonance Energy Transfer Shusheng Zhang*, Yameng Yan, and Sai Bi* Key Laboratory of Eco-chemical Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 0, China 1

2 Table S1. DNA Sequence Used in This Work oligonucleotides name sequences a description DNA aptamer for ATP -TGG AAG GAG GCG TTA TGA amino group modified ATP aptamer with a GGG GGT CCA TTT TTT T spacer end used to fabricate TTT-NH - aptamer-mnps thiol modified DNA -AGT AGG TCA CTT TGT AT-(CH ) -SH- thiolated DNA used to conjugate with LumAuNPs (LumAuNP-DNA) acted as the donor in CRET-BMBPs fluorescein labeled DNA (F-DNA) -F-TGG TTG AAC TAG TTG AA- fluorescein labeled DNA (F-DNA) acted as the acceptor in CRET-BMBPs linker DNA -CGC CTC CTT CCA TTC AAC link LumAuNP-DNA and F-DNA for BMBP TAG TTC AAC CAC GAC ATA CAA CRET-BMBPs detection AGT GAC CTA CT- two-base mismatch linker DNA -CGC CTC CTT CCA TTC ATC TAG TTC AAC CAC GAC ATA CAA These two sequences are used to support the mechanism of CRET AGT GCC CTA CT- noncomplementary linker DNA -CGC CTC CTT CCA AAG TTG ATC AAG TTG GTG CTG TAT GTT TCA CTG GAT GA- thiol modified DNA -SH-(CH ) -AAA AAA GAA GAA TTG AGC- thiolated DNA used to conjugate with LumAuNPs (LumAuNP-DNA) acted as the donor in CRET-TMBPs TMBP fluorescein labeled -F-TCT TTT TTC TTC TT- fluorescein labeled DNA (F-DNA) acted as DNA (F-DNA) the acceptor in CRET-TMBPs biotin labeled DNA -CGC CTC CTT CCA GCT CAA TTC TTC TTT TTT CT-biotin- biotin labeled DNA used to conjugate with streptavidin-hrp acted as the catalyst in CRET-TMBPs inva-1 -TGG AAG GAG GCG- invader DNA were used to accelate the rate invader inva- -TGG AAG GAG G- of ATP detection inva- -TGG AAG GA- inva- -TGG AAG- a The mismatched bases in DNA sequences are marked out with italic and underlined.

3 Characterization of LumAuNPs. The LumAuNPs synthesized by different amounts of luminol solution were charactered with TEM (Figure S1), and UV-visible and fluorescence spectrum was also recorded (Figure S). Figure S1. TEM images of AuNPs synthesized with varying amounts of luminol solution: (a) 1.; (b) 1.; (c).; (d). ml. The corresponding average diameter calculated from a-d are, 1, 1 and nm, respectively. Figure S. UV-visible absorption spectra (left) and fluorescence spectra (right) of AuNPs synthesized by varying amounts of luminol solution: 0 ml of HAuCl solution (0.01%, w/w) 1 was reacted with 1. (black curve), 1. (red curve),. (green curve) and. (blue curve) luminol 1 solution (1.0 - M). 1

4 1 Characterization of Carboxyl-Modified MNPs. The TEM image of the carboxylated MNPs with an average diameter of 1.0 µm is shown in Figure S. Figure S. SEM image of carboxyl-modified MNPs (average diameter of 1.0 µm). The MNPs sample was stored with PBS buffer at oc and ultrasonicated for min prior to each experiment. HPLC Analysis. HPLC analyses were carried out on a Hypersil BDS C1,. 0 mm, µm column using a Waters system comprising a model 00 controller, a model 00 pump, a Rheodyne i manual injector, and a Water photodiode array (PDA) detector. The mobile phase consisted of 0 mm NaHPO and NaHPO phosphate buffer (ph.), degassed by vacuum extraction for min. Samples were injected in µl volumn and eluted at a flow rate of 0. 1 ml/min at oc. The eluate was monitored continuously using an UV absorbance detector at 1 nm. 1 Determination of ATP by HPLC. ATP samples were determined by HPLC performed on Waters 1 system according to the method as described previoiusly. The absorbance peak of ATP was 1 quantified by comparing the retention time and peak height with a known ATP standard. The 1 D-HPLC chromatograms of ATP and the calibration curve for the determination of ATP standard 1 was shown in Figure S. The peak area was linear with the concentration of ATP ranging from 1.0

5 - to.0 - M. The correlation coefficient was 0. and the regression equation was y = 0.x.0 (x was the concentration of ATP, - M; y was the peak height, AU).

6 Figure S. (A) D-HPLC chromatograms of ATP determination. (B) UV/Vis spectra of ATP with a maximun absorption wavelength at nm. (C) HPLC chromatograms of 0 mg/ml ATP at nm with a retention time of about. min. ATP was well resolved and free from interference peaks. The identity of the chromatographic peak was confirmed not only by its retention time but also by its spectrum. (D) The calibration curve for the determination of ATP by HPLC quantified at nm. The concentration of ATP are 0, 1.0 -,.0 -, 1.0 -,.0 -,.0 -, and.0 - M. Effect of Fluorescein Concentration on Luminol-H O -HRP-fluorescein CRET System. In this experiment, free fluorescein was used to investigate the effect of fluorescein concentration on luminol-h O -HRP-fluorescein CRET system. From Figure S, it is clear that with the increasing 1 of fluorescein concentration, the total emission between 0 nm and 00 nm is enhanced and the 1 intensity at nm increases while that at nm decreases. The enhancement of energy transfer 1 with fluorescein concentration can be ascribed to an increase of overlap between donor 1 chemiluminescence and acceptor absorption. Furthermore, depending on the fluorescein 1 1 concentration, emission from the acceptor occured a slight red-shift with wavelengths varying from to 0 nm which can be attributed to a concentration effect. [1] 1

7 Figure S. CL spectra of luminol-h O -HRP-fluorescein system against wavelengths of emission at different concentrations of fluorescein. The concentrations of fluorescein are listed in the figure. Synthesis of CitAuNPs and Au@Fe O. AuNPs used for the following preparation of magnetic Fe O -Au core-shell nanoparticles (Au@Fe O ) were synthesized by following the method of reduction of tetrachloroauric acid with trisodium citrate which has been carried out by Ambrosi et al. [] Briefly, 0 ml of 0.0% HAuCl solution was boiled with vigorous stirring, and then. ml of 1% trisodium citrate solution was added dropwise to the boiling solution quickly. When the color of the solution turned from gray yellow to deep red, it could indicate the formation of AuNPs. After cooling down the resulting colloidal suspension to room temperature with stirring, the colloidal AuNPs were obtained. And then, the synthesized AuNPs were capped on the surface of 1 NH -MNPs through Au-N bonds to obtain the Au@Fe O. Briefly, ml of the CitAuNPs solution 1 (~ nm) was added to 0 µl of mg/ml NH -MNPs. After shaking gently for h at room 1 temperature, the Au@Fe O was obtained by washing with 00 µl of 0.01 M ph. phosphate 1 buffer containing 0. M NaCl for three times,, and then redispersed in 00 µl of 0.01 M ph. 1 phosphate buffer containing 0. M NaCl. 1 1 The Control Experiment to Support the Mechanism of CRET. 1 Methods. After LumAuNP-DNA and F-DNA incubating with Au@Fe O for min, 0 µl of nm linker (complementary sequence, two-bases mismatched sequences and non 1 complementary sequences) in PBS buffer solution was added and incubated for 0 min to hybridization. In order to ensure that all the ssdna were adsorbed on the Fe O -Au core-shell

8 nanoparticles, an excess O were applied. After magnetic separation, 0 µl of the supernatant was transferred to the quartz cuvette containing 0 µl of 1.0 µm HRP. The CL measurements were performed with a BPCL ultraweak luminescence analyzer (Institute of Biophysics Academic Sinica, Beijing, China). The CL reaction was triggered by injecting 00 µl of. - M H O with a syringe through a septum after the CL analyzer began to record at s. The kinetics of CL signals between 0 to 0 s were recorded and the peak heights of the emission curves were measured by means of a photon counting unit. Results and Discussion. A control experiment to support the CRET mechanism was carried out by employing 1.0 nm linker DNA with two-bases mismatched DNA sequences and non cdna sequences compared with complementary linker sequence. However, in this experiment the CL detection was obscured by a high background. This can be explained that although the distances 1 were not suitable for the CRET when LumAuNP-DNA and F-DNA were dispersed in solution 1 freely, and the CL intensities were indeedly increased after these two probes hybridized with the 1 linker DNA based on CRET, however, it was difficult to ensure these two probes could not occur 1 CL radomly for CRET system in which lunimol was acted as the donor and fluorescein was acted 1 as the acceptor and enhancer. In order to circumvent this problem, we synthesized magnetic 1 Au@Fe O to overcome the high background of CRET-BMBPs based on the different electrostatic 1 propensities of single- and double-stranded oligonucleotides (dsdna and ssdna) adsorption on 1 gold nanoparticles. The principle of the improved proposal is illustrated in Scheme S1. The TEM 0 images of amino-modified MNPs, CitAuNPs, and Au@ Fe O were recorded (Figure S).

9 Scheme S1. Schematics of the preparation of O through Au-N bonds (upper part), and the assay procedure of BMBPs based on CRET (lower part). Firstly, LumAuNP-DNA and F-DNA are incubated with O for min. In the absence of linker DNA, two kinds of ssdna adsorb on O by electrostatic interaction. Upon addding the linker DNA, ssdna are forced to dissociate from the surface of O to hybridy with linker DNA. After separating the O from the solution, 0 µl of supernatant is transferred into the determination cuvette which contains 0 µl of 1.0 µm HRP. The CL detection is triggered by injecting 00 µl of. - M H O. 1 Figure S. TEM images of (A) amino-modified MNPs with the average size of.0 µm, (B) AuNPs 1 with an average diameter of nm, and (C) the surface of an Au@Fe O nanoparticles. From (C), 1 the gold nanoparticles cover the whole surface of the MNPs and retain their original sizes. 1 In this study, Au@Fe O not only serves as potential adsorption carriers of ssdna to decrease

10 the background, but also simplify the separation procedure of the analysis process. As shown in Figure S, a well-defined CL signal was obtianed for the complementary linker DNA, and no response was detected for the non-cdna linker sequences which was equivalent to that of the background (without linker DNA in the system). The sequence specificity of this BMBPs system was also investigated by using one-base mismatched linker sequences for LumAuNP-DNA and F-DNA simultaneously. It was found that the signal was significantly weaker than that of the complementary squences which was not significantly different from the background. We proposed that this increased sequence specificity is attributed to the solution-phase BMBPs employed in this work. For the solid-phase counterpart, it often showed limited ability for the discrimination of target DNA mismatches, which possibly arises from the surface-induced destabilization effect on the duplex hybridization. [] The results proved that the CRET-BMBPs in this study with high 1 selectivity could be used to identify DNA sequence for target DNA hybridizaiton. 1 1 Figure S. CL intensity versus different linker DNA sequences after hybridization with 1 LumAuNP-DNA and F-DNA. Each data was obtained from three independent measurements. The 1 concentration of DNA is 1.0 nm. 1 1 Parameters Optimization for ATP Analysis by CRET-BMBPs and CRET-TMBPs. Incubation

11 temperature and reaction time for the ATP and aptamers are the most two important parameters to optimize the analysis system. A series of temperture-changing CRET assays ( o C, o C, o C) were carried out in the absence and presence of ATP for 0 min. As observed from Figure. S and S, in the absence of ATP, the CL intensities were increased with raising the temperature from o C to o C which can be attributed to the denaturation of the DNA duplex assembly with the temperature rising, and less and less BMBPs and TMBPs were hybridized with the aptamer immoblized on the MNPs surface. However, BMBPs and TMBPs were fabricated through hybridization of ssdna or triplex DNA binder, thus with increasing the temperature the stuctures of BMBPs and TMBPs were disassembled that were not favorable to CRET. Consequently, the CL intensities were not increase significantly with the temperature increasing. The similar CL intensities of BMBPs at o C and o C can be explained as considering the melting temperature 1 (Tm) of these short synthetic strands of ~0 o C, [] the sturcure of BMBPs disassembled from the 1 MNPs at o C should be destroyed more completely than at o C. Although the strucure of 1 BMBPs was destroyed almost completely at o C which could not favorable to CRET, the 1 amounts of each labeled DNA sequence (LumAuNP-DNA and F-DNA) at o C were much more 1 than that at o C. Thus, CL signals for BMBPs at o C were not different from that at o C. The 1 situation was a slightly different for the TMBPs because the TMBPs were fabricated through a 1 strong triplex binder, BePI, which Tm- and Tm-1 (Tm- and Tm-1 are represented a melting 1 transiton from a triplex to a duplex, and a duplex to ssdna, respectively) are. and 1. o C, 0 respectively. Thus, with increasing the temperature, the CL signals at o C were slightly higher 1 than that at o C owing to the contribution of BePI because TMBPs were destroyed completely at o C. In the presence of ATP, for the BMBPs, a maximal intensities was reached at ~0 min when

12 the temperature was at o C, and more ripid at o C (~1 min) and o C (~min). However, considering the duplex DNA dehybridizaiton at higher temperature, thus the efficiency of CRET-BMBPs at and o C were not higher than o C which resulted in a lower CL intensities. For the TMBPs, due to the assistance of triplex binder, the CL intensites had a slightly effect at o C and achieved a rapid maximum at ~1 min compared with ~ min at o C. Because the binding efficiency of BePI was much more weakened at o C, thus, ssdna were free in the solution which was not favorable for CRET, and the CL intensites were similar to that of in the absence of ATP. The observations can also be considered as the evidence of structure switching. [] This can be explained as the aptamer-target complex was not directly formed between the target and the free target-binding site of the aptamer but rather between the target and the aptamer that was patially 1 hybridized by DNA and further blocked the binding site of an aptamer. As a result, the affinity of 1 an aptamer for its target reduced. At low temperature, because most of the DNA aptamer 1 molecules existed in the duplex structure the rapid structure switching did not occur, and the ATP 1 binding site was occupied by linker DNA partially. Whereas at higher temperature, a rapid 1 structural transition happened because more CRET probes were forced to dissociate from the 1 duplex assembly, and as a result, more aptamer molecules had their ATP binding site freed for ATP 1 binding. Considering both of the melting temperature (Tm) of DNA hybridizaiotn and the 1 temperature of ATP/aptamer binding, we chose o C for further assays of the system, although the 0 time required for the maximal CL intensity was longer than at higher temperature. 1 In order to circumvent the slow response problem, an invader DNA was added to help release the CRET probes and accelerate the rate of aptamer structure-switching process. The improved 1

13 strategy is depicted in Scheme S and the results are detailed discussed in the manuscript. Considering both the rate of release and the level of background, inva-, a -mer invader DNA, was selected as tradeoff for the subsequently analytical work. Scheme S. Schematics of CRET-BMBPs (A) and CRET-TMBPs (B) for the detection of ATP by employing invader. The rate of aptamer structure-switching process can be significantly increased by adding invader. The invader sequences are listed in Table S1 and other sequences are the same as shown in Scheme in the main text. 1 Figure S. Temperature effects on CRET-BMBPs at o C, o C and o C. 1

14 Figure S. Temperature effects on CRET-TMBPs at o C, o C and o C. Comparative Study between the Present Proposals and HPLC Figure S. The correlation of results for the determination of ATP in cancer cells by HPLC (x axis) and CRET-BMBPs (A) and CRET-TMBPs (B) (y axis) with correlation coefficients of 0. and 0., respectively. References (1) Díaz, A. N.; García, J. A. G.; Lovillo, J. J. Biolumin. Chemilumin. 1, 1, () Ambrosi,A.; Castañeda, M. T.; Killard, A. J.; Smyth, M. R.; Alegret, S.; Merkoci, A. Anal. 1 Chem. 00,, () Rupcich, N.; Nutiu, R.; Li, Y.; Brennan, J. D. Anal. Chem. 00,,

15 () Nutiu, R.; Li, Y. J. Am. Chem. Soc. 00, 1, 1-. 1