Hairpin-Contained i-Motif Based Fluorescent Ratiometric Probe for

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Hairpin-Contained i-Motif Based Fluorescent Ratiometric Probe for High-Resolution and Sensitive Response of Small pH Variations Wenjie Ma, Lv'an Yan, Xiaoxiao He, Taiping Qing, Yanli Lei, Zhenzhen Qiao, Dinggeng He, Kaihang Huang, and Kemin Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03972 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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Analytical Chemistry

Hairpin-Contained i-Motif Based Fluorescent Ratiometric Probe for High-Resolution and Sensitive Response of Small pH Variations Wenjie Ma, Lv’an Yan, Xiaoxiao He∗, Taiping Qing, Yanli Lei, Zhenzhen Qiao, Dinggeng He, Kaihang Huang, Kemin Wang∗ State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, Changsha 410082, China ABSTRACT: Intracellular pH (pHi) is an important parameter associated with cellular behaviors and pathological conditions. Sensing pHi and monitoring its changes are essential but challenging due to the lack of high-sensitive probes. Herein, a ratiometric fluorescent probe with ultra pH-sensitivity is developed based on hairpin-contained i-motif strand (I-strand, labeled with Rhodamine Green and BHQ2 at two termini) and complementary strand (C-strand, labeled with Rhodamine Red at its 5'-end). At neutral pH, both I-strand and C-strand hybridize into a rigid duplex (I-C), which holds the Rhodamine Red and the BHQ2 in close proximity. As a result, the fluorescence emission (F597 nm) of the Rhodamine Red is strongly suppressed, while the Rhodamine Green (F542 nm) is in a “signal on” state. However, the slightly acidic pH enforced the I-strand to form an intramolecular i-motif and initiated the dehybridization of I-C duplex, leading to Rhodamine Red in a "signal on" state and a decreased fluorescence of Rhodamine Green. The ratio (F542 nm/F597 nm) can be used as a signal for pH sensing. Due to the rational internal hairpin design of IC duplex probe, almost 70-fold change in the ratio was observed in the physiological pH range (6.50-7.40). This probe possesses efficient stability, fast response and reversible pH measurement capabilities. Furthermore, intracellular application of the ratiometric probe was demonstrated on the example of SMMC-7721 cells. With different recognition elements in engineering of imotif based platforms, the design might hold great potential to become a versatile strategy for intracellular pH sensing.

pH is a pivotal physiological parameter that plays a vital role in the regulation of various cellular behaviors and biological processes, including cell proliferation and apoptosis, cellular metabolism, neuronal activity, inflammation and multidrug resistance.1-6 The abnormal pH is working as a typical hallmark of many cardiopulmonary and neurologic problems, such as myocardial ischemia7 and Alzheimer’s.8 Therefore, monitoring and sensing pH gradient in living organisms is critically significant for learning cellular functions and gaining a better understanding of physiological and pathological processes. A variety of techniques have been established for pH sensing in biomedical applications, such as H+-permeable microelectrodes,9 nuclear magnetic resonance (NMR) spectroscopy10 and optical microscopy.11 However, the most of mentioned techniques require sophisticated instrumentations and complicated sample preparation procedures, which limit their wider applications. In contrast, fluorescent spectrometry, based on pH-induced alteration in fluorescence intensity, has attracted considerable interest to address the growing requirements for rapid and sensitive pH detection because of the advantages of the unrivaled spatiotemporal resolution, rapid response time, high signal-to-noise ratio and noninvasiveness.12-15 Unfortunately, one potential drawback for most of these fluorescent strategies is their broad pH response which respond over a fixed pH window that typically spans 1.5-2.0 pH units.16 It is well known that the cellular

homeostasis is tightly regulated by relatively small changes in intracellular pH. For example, the intracellular pH (pHi) change range of tumour cells (7.12-7.65) and normal tissues (6.99-7.20) are only 0.2-0.4 pH units; the acidic interstitial extracellular pH variation range (pHe) are 0.3-0.4 pH units (6.2-6.9 compared with 7.3-7.4).3, 17 As a consequence, this lack of sharp pH response makes it difficult to detect subtle pH changes in intracellular organelles. The narrowing of response sensitivity is even more challenging. To date, a series of new sensing strategies have been reported to improve the sensitivity of pH detection, which do deliver sharp response sensitivity.18-20 For example, Gao et al. reported a series of ultra-pH sensitive (UPS) block copolymers, which displayed a sharp on/off pH response for amplifying tumour microenvironmental signals.19 However, the laborious and complicated synthesis, modification steps for most of polymeric sensors have limited this progress. Hence, it is of great value to develop novel fluorescent sensors for pH quantification with sharp response, high resolution, and ease of synthesis. Alternatively, DNA nanostructures, with pHresponsive ability, have been widely used in the field of biomedical due to their excellent biocompatibility, automated synthesis, specific base pairing interactions, versatility and programmability.21 Various DNA nanostructures have been tried, including intermolecular triplex DNA,22, 23 t-switch,24

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DNA tweezers,25 A-motif 26 and i-motif.27-34 Among numerous of DNA nanostructures, the i-motif structures have attracted wide interest of researchers, as they can be switched flexibility and offer access to new types of materials. The i-motif, a fourstranded DNA structure formed from the cytosine (C)-rich ssDNA sequences, has emerged as a versatile pH-switchable sequence for functional DNA structure, which consists of two parallel duplexes maintained by C·CH+ pairs intercalated with each other in an antiparallel orientation. Up to now, various pH-activated fluorescent probes based on DNA i-motif structure have been proposed for pH monitoring inside living cells17 and living organisms.20 Especially, Nesterova et al. reported design principles of highly responsive pH sensors based on DNA i-motif.30 Both the response sensitivity and transition midpoint can be tuned via rational manipulations of an i-motif structure as well as incorporation of allosteric control elements. After incorporated an internal hairpin, the new probe showed higher values for the Hill coefficient n, and narrower ∆pH10-90 compared to the parent i-motif. In contrast to the effect of an internal hairpin, incorporation of an external hairpin showed an opposite effect: a decrease in the i-motif folding cooperativity. It provides a favorable foundation for the reasonable design and widespread use of i-motif for pH sensors. Nevertheless, this strategy may have signal fluctuations and exhibit high-background signals because of the single emission intensity changes, also it will inevitably lead to degradation of the reliability of the measured results. More robust signals acquired by twowavelength ratiometric methods which can permit simultaneous recording of the relative changes of two separated wavelengths instead of measuring single emission intensity changes. Ratiometric fluorescence measurements can avoid the influence of environmental effects and overcome the shortcomings in the single emission intensity examinations through ratiometric self-calibration of the two emission peaks effectively, thus enhancing the accuracy of measurements. With this in mind, we herein designed an i-motif based fluorescent ratiometric probe for high-resolution and sensitive response of small pH variations by embedding an internal hairpin and a complementary strand. As illustrated in Scheme 1, the design consists of two single-stranded DNA, one is a 39 mer single-stranded oligonucleotide with an internal hairpin (named as I-strand), functionalized with a fluorophore (F2=Rhodamine Green) and a quencher (Q=BHQ2) at its 5' and 3' termini, respectively. The other one is C-strand, a short single-stranded DNA which is complementary to i-motif strand partially, labeled with another fluorophore (F1=Rhodamine Red) at the 5'-end. At neutral pH, both two strands hybridize into a rigid duplex (I-C), in such a way that F1 is kept in close proximity to Q and thus causing quenching of the F1 emission, while the F2 is in a “signal on” state. At acidic pH, the cytosine nucleobases get partially protonated and the signal states of the two fluorophores exchanged because of the formation of i-motif structure. The fluorescence intensity of the I-C duplex probe at the two emissions is pHdependent simultaneously. The fluorescence emission ratio (F542 nm/F597 nm) can be used as a signal readout model for quantitation of pH values. The new fabricated fluorescent ratiometric probe by i-motif with an internal hairpin displays high-resolution and sensitive response for small pH variations.

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Scheme 1. Schematic representation of pH-driven fluorescent ratiometric probe. I-strand represents the i-motif formation strand with an internal hairpin. C-strand represents the complementary strand of I-strand with Rhodamine Red

EXPERIMENTAL SECTION Chemicals and Reagents. All the DNA sequences used in this work were synthesized by TAKARA Biotechnology Inc. (Dalian, China) and purified by reverse-phase high performance liquid chromatography (HPLC). Sequences of oligonucleotides are listed in Table S1 (Supporting Information). The solutions of phosphate buffer saline (PBS, 10 mM) at the corresponding pH were prepared using a mixture of 0.1 M NaH2PO4·2H2O and 0.1 M Na2HPO4·12H2O in different ratio, and added a certain amount of NaCl and KCl. The pH of all solutions was adjusted slightly with 0.5 M HCl and 0.5 M NaOH. Cell medium RPMI 1640 was purchased from Clontech (Mountain View, CA, USA). Fetal bovine serum was acquired from Hyclone (Logan, UT, USA). 35 mm glass bottom dishes were purchased from MatTek (Ashland, MA, USA). SMMC-7721 cells (human hepatocellular cancer) were obtained from Cell Bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences. Deionized water was obtained by the Milli-Q ultrapure water system (Barnstead/Thermolyne NANO pure, Dubuque, IA, USA). All other reagents were of the highest grade without further purification. Apparatus. UV-vis absorption spectra were collected using a Biospec-nano UV-vis spectrophot ometer (Kyoto, Japan). Circular dichroism (CD) spectra were acquired on a Bio-Logic MOS-500 CD spectrophotometer (Claix, France). The fluorescent spectra were collected using a Hitachi F-7000 fluorescence spectrometer (Tokyo, Japan) equipped with a temperature controller. The confocal microscope images were acquired on a FV500 confocal microscope (Olympus, Japan). Cells were maintained at 37 °C in a 5% CO2 atmosphere in humidified HF90 CO2 incubator (Shanghai Lishen Scientific Equipment Co. Ltd.). The fluorescence imaging was taken by an IVIS Lumina II in vivo imaging system (Caliper Life Sicence, U.S.A.). All the pH measurements of buffer were acquired on Thermo Scientific Orion 3 Star pH-meter (Waltham, MA, USA) and a miniature pH meter (HACA, H130 miniiab, USA). UV-vis Spectrum Measurement. The I-C duplex probe was diluted to a certain concentration of 500 nM in 10 mM phosphate-buffered saline (PBS) with different pH. UV-vis spectra were recorded in the 220-320 nm range with a data interval of 1 nm and processed using Sigma Plot software. For thermodynamic stability, 500 nM i-motif structure (pH 6.50) and I-C duplex structure (pH 7.40) were used to investigate


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Analytical Chemistry the thermal profiles at different temperature. The changes at 295 nm were used to indicate the unfolding of i-motif structure. Changes at 260 nm were used to indicate the melting of duplex probe. CD Measurement. Circular dichroism (CD) spectra were carried out in the range of 220-340 nm at 25 °C in a quartz cuvette. A background CD spectrum of corresponding buffer solution was subtracted from the average scan for each sample. Fluorescence Analysis. The I-C duplex probe was diluted to a concentration of 500 nM. Then the fluorescent spectra was measured after incubated at 25 °C for 30 min in different PBS buffer (pH 5.60-8.00) to characterize its pH response performance. Emission spectra of Rhodamine Green were collected from 510 nm to 700 nm in 1 nm increments while excited at 498 nm. The emission spectra of Rhodamine Red were collected from 580 nm to 700 nm in 1 nm increments while excited at 560 nm. Both excitation and emission slits were set at 5 nm. PMT voltages of Rhodamine Green and Red channels were 700 V and 950 V, respectively. For the reversibility and real-time of fluorescence scanning of I-C duplex probe, the concentration of probes was 100 nM. The fluorescence imaging analyses of I-C duplex probe was taken by an IVIS Lumina II in vivo imaging system. Rhodamine Green was excited at 465 nm and the fluorescence emission filter was GFP, whereas Rhodamine Red was excited at 535 nm and the fluorescence emission filter was DsRed. The fluorescence images and pseudoimages were presented after processing by software Image Proplus 6.0. Cell Culture and Fixation. SMMC-7721 (human hepatocellular cancer) cells were cultured in RPMI 1640 medium supplemented with 13% fetal bovine serum (FBS) and 100 U/mL penicillin-gentamicin. Cells were seeded on 35mm glass bottom dishes and incubated at 37 °C in a humidified incubator containing 5% wt/vol CO2 for 1-2 days prior to treatment. After removal of the medium, the cells were first washed three times with phosphate buffered saline (PBS, pH 7.40, calcium and magnesium free) and then fixed on the culture dishes with fixing solution (the volume ratio of methanol to acetic acid was 3:1) for 8 min at room temperature followed by three PBS washes. Confocal Fluorescence Microscopy Imaging. The I-C duplex probe was first fabricated under alkaline conditions (PBS, pH 7.40). The culture dishes with fixed cells were incubated with clear media containing 200 nM I-C duplex probes for 3 h. And then, all cells were washed to remove excess probes by different pH buffer (pH 5.60, 6.50, 7.00, and 7.40) and incubated with 200 µL PBS with corresponding pH values for 30 min to regulate the intracellular pH. Fluorescent images were acquired on a laser scanning confocal microscope with a 100× oil immersion objective. Excitation wavelength and emission filters were described as follows. Rhodamine Green: Ex = 488 nm, Em = 505-560 nm bandpass; Rhodamine Red: Ex = 543 nm, Em = 560 nm long-pass.

RESULTS AND DISCUSSION Characterization of i-motif Formation in Slightly Acidic pH. As an i-motif formation strand, the I-strand presents a sharp pH transition between the “closed” and “open” states in different pH values. First, the pH-dependent structural transformation of the I-strand from random coil to i-motif

structure was studied by UV-vis absorption in various pH solutions. As shown in Figure 1A, with the decrease of the pH value, a gradual increase of UV-absorption at 295 nm (characteristic peaks of i-motif) was observed. It suggested that the I-strand could undergo a conformational change from random coil to i-motif structure under acidic conditions, which agreed with the previous report.35 In order to investigate whether the addition of the complementary strand would affect the formation of the i-motif structure, we further carried out the UV spectra of the I-C duplex in different pH solutions. As we can see, it showed the similar behavior in different pH solutions, indicating that the I-C duplex could also undergo a structural change driven by a pH change (Figure 1B).

Figure 1. UV absorption spectra of I-strand (A) and I-C duplex (B) in 10 mM PBS buffer at different pH values. The concentration of the I-strand and I-C duplex was both 500 nM.

The other common method used to prove i-motif formation is to monitor the change of circular dischroism (CD) spectra. So, the pH-value-stimulated transitions between random coil and i-motif structures were further examined by CD spectra at different pH. As shown in Figure S1, both the I-strand and I-C duplex exhibited the same CD phenomenon under various pH values. With the pH changed from 8.00 to pH 5.60, the positive band near 275 nm and the negative band near 245 nm were red-shifted to 290 nm and 260 nm, respectively. This result could be explained by the polynucleotide helicity and base stacking of unstructured ssDNA. These two dominant peaks near 290 nm and 260 nm in the corresponding CD spectra, are agreed with the characteristics of i-motif structures as reported previously. 28, 31 These CD results suggested that the pH-driven conformational alteration of i-motif sequence went from the random coil to i-motif structure regardless of whether the complementary strand exists, which provided strong evidence that the i-motif tetraplex structure can be well formed under acidic conditions. Thermodynamic Stability. The thermal stability of DNA folded structures is crucial for the explanation of sensing system operation mechanism.34, 36, 37 So, the thermal denaturation profiles of I-strand (pH 6.50) and I-C duplex (pH 7.40) were investigated. The absorption changes at 295 nm (almost exclusively indicative of i-motif structure) were used to monitor the unfolding of i-motif structure.34 The melting transition in the duplex could be simply monitored via absorbance changes at 260 nm (indicative of double-stranded to single-stranded transition).38 As shown in Figure S2, the Tm value of I-strand and I-C duplex strand were 36 °C, 46 °C, respectively. These results indicated that both I-strand probe and I-C duplex probe could existed as the intrinsic states (I was single strand, I-C was double strand), which could change into i-motif structure only after altering pH. In other words,



this ratiometric fluorescent probe for pH sensing could be carried out in experimental conditions (25 °C). Performance of I-C Duplex Probe. After verifying the thermal stability and the i-motif structure of I-C duplex could be formed in slightly acidic condition, the response range and detection capability of this ratiometric probe for pH sensing was investigated. As shown in Figure 2A and 2B, sequential decreases in the fluorescence emission intensity of Rhodamine Green (F542 nm) and gradual increase of Rhodamine Red (F597 nm) were observed with decreasing pH. It was consistent with the change of Rhodamine Green in single I-strand based probe (Figure S3). At acidic pH, the I-strand which contains considerable cytosine bases could fold into the compact, rigid intramolecular i-motif structure through hemi-protonated cytosine-cytosine (C·C+) base pair formation. As a consequence of the changes of conformation, the Rhodamine Green was kept in close proximity to the BHQ2, and the fluorescence was in a “signal off” state due to the proximityinduced energy transfer. For I-C duplex probe, the released Cstrand would be a “signal on” state due to far away from the quencher BHQ2. So, the Rhodamine Green/Rhodamine Red signal serves as a ratiometric signal readout mode, could reduce environmental interference and enable more precise detection. The plot of fluorescent ratios (F542 nm/F597 nm) of Rhodamine Green/Rhodamine Red as a function of pH showed a characteristic sigmoidal increase from pH 5.60 to 8.00, with the sharp transition in the pH range of 6.50-7.40 (Figure 2C). An almost 70-fold change in the ratio (from 0.26 to 18.36) was observed when the value of pH increased from 6.50 to 7.40, a significantly narrow pH range within almost a unit. Such narrow pH response range would be due to the implantation of an internal hairpin.30 It showed an extremely

Figure 2. pH-dependent fluorescence emission spectra of I-C duplex probe from the Rhodamine Green (A) and Rhodamine Red (B) in buffer at pH 5.60, 5.91, 6.18, 6.50, 6.65, 6.80, 7.00, 7.14, 7.30, 7.40, 7.72 and 8.00; (C) Plot of F542/F597 as a function of pH (from 5.60 to 8.00); (D) Linear relationship of the ratiometric fluorescence intensity (F542/F597) versus pH values. The excitation wavelengths of Rhodamine Green and Rhodamine Red were 498 nm and 560 nm, respectively. The concentration of the I-C duplex was 500 nM. Error bars represent variations between three measurements.

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high sensitivity and efficient signal output, which was much higher than the previous reports.14, 28 Surprisingly, compared with the I-strand based probe, the pH response range of I-C duplex probe was extended to 6.50-7.40, with a sharp pH response property. Also of note, this range matches with the pH values of physiological environments, which is in accordance with our postulated principle of the pH-responsive probe. More importantly, it was further verified that the fluorescence intensity of ratios (F542 nm/F597 nm) was linear against pH variation. A pH calibration curve in the PBS buffer solution was depicted in Figure 2D, which presented a good linear correlation (R2 = 0.9863) in the range of 6.50-7.40. We considered that the I-C duplex based ratiometric probe could be appropriate for pH sensing in physiological environment and intracellular pH imaging. Fluorescence change mechanism. To explore the potential mechanism of high-resolution and sensitive response for small pH variations, we further investigated the effect of DNA structure and pH on fluorescent dyes labeled in ratiometric probe. For this purpose, an i-motif formation strand labeled only with Rhodamine Green (named as I′-strand), was specially designed. As described in Figure 3A, the emission characteristics of the Rhodamine Red (labeled in C-strand) is not affected by the changed pH value, which is consistent with previous literature.14 However, the emission characteristics of the Rhodamine Green (labeled in I′-strand) is partial affected by the changed DNA configuration and pH value (from 5.60 to 8.00), which is not agree with that the fluorophore of Rhodamine Green is insensitive to pH from 4.0 to 9.0 (Figure 3B). 30, 39, 40 Therefore, we considered whether the formation of i-motif structure could affect the fluorescence emission characteristics of Rhodamine Green in our detection system. After that, two non i-motif formation strands (named as N1strand and N2-strand) labeled with Rhodamine Green were selected to study their pH stability. As expected, the fluorescence intensity of the Rhodamine Green is insensitive to pH variations in these non i-motif sequences (Figure S4). In view of this situation, we then selected two other wellcharacterized i-motif sequences (named as I1-strand and I2strand) labeled with Rhodamine Green. It is reported that these sequences labeled with Rhodamine Green are insensitive to different pH.14, 30 Unexpectedly, the fluorescence intensity of Rhodamine Green in these i-motif sequences also has slight variation at different pH, liked as I′-strand in this sensing system (Figure S5). In other words, an i-motif, formed from

Figure 3. Effect of pH value on the fluorescence intensity of Rhodamine Red (A) and Rhodamine Green (B). The Rhodamine Red is labeled in C-strand and Rhodamine green is labeled in I′strand. The concentration of the C-strand and I′-strand was both 500 nM.


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Analytical Chemistry cytidine-rich DNA fragments that fold into a H+-mediated quadruplex, can reduce the fluorescence intensity of Rhodamine Green to a certain extent. There results indicated that the emission characteristic of the Rhodamine Green is sensitive to the i-motif structure but not the pH variations. As a result, the signal output of the proposed ratiometric probe can be generated from both i-motif structure and pH-mediated conformational change. The i-motif structure in I-C duplex could further facilitate the fluorescence quenching, leading to higher detection sensitivity. Characteristics of I-C Duplex Probe. To our best knowledge, the intracellular environment is absolutely different from the pure buffer solution. In order to testify its universality, we investigated the detection capability of the I-C duplex probe with different concentrations. The I-C duplex probe can respond well to changes in pH in different probe concentrations (Figure S6). By adding different concentrations (100-500 nM) of the I-C duplex probes into pH 7.00 solutions, we observed an increase in fluorescent intensities for both Rhodamine Green and Rhodamine Red. However, the ratios of both Rhodamine Green and Rhodamine Red were nearly constant at about 10.62-fold, independent of the probe concentrations (Figure 4). These results guaranteed its accuracy of the pH probe in different detection condition.

Figure 4. (A) The fluorescence images of Rhodamine Green (green) and Rhodamine Red channels (red) and resulting ratio of Rhodamine Green/ Rhodamine Red (ratio, mixed colors) of wells containing different concentrations of I-C duplex probes. Rhodamine Green was excited at 465 nm and the fluorescence emission filter was GFP, whereas Rhodamine Red was excited at 535 nm and the fluorescence emission filter was DsRed. The ratiometric images of green to red fluorescence intensity of each well obtained from software Image Proplus 6.0. (B) Values and ratios of fluorescence intensities of Rhodamine Green at 542 nm and Rhodamine Red at 597 nm in different probe concentrations.

Moreover, to see if the I-C duplex probe could switch reversibly between random coil and quadruplex structure upon variation of pH, the fluorescence reversibility against pH was carried out by adjusting the pH value between 7.60 and 6.40 repeatedly. As described in Figure 5A, the ratio of fluorescence intensity (F542 nm/F597 nm) of the I-C duplex probe showed a good reproducibility between the weak alkaline solution (pH 7.60) and the weak acidic solution (pH 6.40). Evidently, a reversible operation of the I-C duplex probe can be triggered by the changes of the solution pH. As expected, it showed excellent reversibility for several pH cycles without observable degradation in different pH solutions, with negligible change in efficiency. Moreover, the results above clearly showed that the conformation of I-C duplex probe can change flexibly under different pH values. In order to further

elucidate the conformational dynamics of i-motif structure, we monitored the real-time changes of I-C duplex probe in fluorescence emission at 542 nm and 597 nm after the alternate addition of HCl and NaOH. As we can see, changes in the pH environment lead to fast changes in the fluorescence intensity of the two fluorescent dyes (Figure 5B and 5C). These results showed that this probe has quick response ability after undergoing pH variation. Therefore, this ratiometric pH probe is expected to apply to detect intracellular pH changes associated with biological processes due to its concentrationsindependent, reversibility, fast response, and physiological pH response ranges.

Figure 5. (A) The reversibility of the ratiometric probe against pH change between high (7.60) and low (6.40), repeatedly. (B) and (C) The real-time of fluorescence changes of I-C duplex probe after addition of acid (H+) and base (OH-). The arrow marks the addition of H+ or OH-. The emission of Rhodamine Green and Rhodamine Red were 542 nm and 597 nm, respectively. The concentration of the I-C duplex probe was 100 nM.

Bioimaging Applications. The excellent reversibility and high sensitivity in physiological pH stimulated us to further investigate stimuli-responsive behavior of the ratiometric probe in cancer cell system (SMMC-7721 cancer cell was selected as a model). As we can see in Figure 6A, the fluorescence intensity of the Rhodamine Green (green channel) in cells decreased with pH decrease from pH 7.40 to pH 5.60, while that of Rhodamine Red (red channel) was increased gradually. At weak acidic condition, the fluorescence of Rhodamine Red seems to locate in cytomembrane, which was possibly because of the G-riched C-strand trend to bind to the cells surface protein.41-43 The merge graph of channel green and channel red presented an obvious color change along with the pH variation. By calculating the average fluorescence intensity of green channel and red channel using ImageJ software, the ratiometric imaging of green to red (Fgreen/Fred) displayed a characteristic pH-dependent signal. The fluorescence intensity ratio (Fgreen/Fred) was linear against pH values from 5.60 to 7.40 in SMMC-7721 cancer cells (Figure 6B). Comparing with a cell free and cell systems, we found that both of the calibration curves possessed the same lineal trends but showed a little difference due to the distinct



working environmental conditions and experimental instruments. Based on this ratiometric calibration curve, the pH values of the SMMC-7721 cancer cells could be determined with high resolution.

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signal output and high sensitivity, was achieved. With different recognition elements in engineering the probe, the design might hold great potential as a versatile strategy for intracellular pH sensing.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional information as noted in text: Oligonucleotides used in the work, CD spectra of the I-strand and I-C duplex, thermodynamic stability of I-strand and I-C duplex, the pH-dependent fluorescence emission spectra of Istrand, stability of Rhodamine Green in different non imotif and i-motif sequences in different pH. Sensing properties of I-C duplex in different probe concentrations. (PDF)

AUTHOR INFORMATION Corresponding Author * Tel: 86-731-88821566; Fax: 86-731-88821566; E-mail: [email protected]. * Tel: 86-731-88821566; Fax: 86-731-88821566; E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Figure 6. Ratiometric calibration of pH in fixed SMMC-7721 cancer cells. (A) Confocal fluorescence imaging of ratiometric probe exposed to external media at pH 5.60, 6.50, 7.00, and 7.40, respectively. The excitation wavelength of Rhodamine Green was 488 nm, and the images were collected in the ranges of 505-560 nm (first row). The excitation wavelength of Rhodamine Red was 543 nm, and the images were collected >560 nm (second row). The corresponding merge images (third row) and the fluorescence intensity of each cell were obtained from green (505-560 nm) and red (>560 nm) channels by software Image Proplus 6.0 (fourth row). (B) Calibration curve of intracellular pH sensing.

CONCLUSION In summary, we have presented a novel ratiometric i-motif probe for high-resolution and sensitive response of small pH variations. The probe possesses abilities of efficient stability, fast response, concentrations-independent detection, and reversible pH measurement. The ratiometric sensing strategy can minimize complex biological environments effects and enable more precise detection. Taking unparalleled advantage of the conformation changes of i-motif and the implantation of an internal hairpin, the ratiometric probe showed a significantly narrow pH response range (0.90 units). After added a complementary strand, the pH response range of I-C duplex based ratiometric probe was extended to 6.50-7.40, which matched with the pH values of physiological conditions. Furthermore, a successful application of this ratiometric fluorescent probe to measure pH in cancer cells, with efficient

This work was supported in part by the Key Project of Natural Science Foundation of China (Grants 21675046 and 21521063). The key point research and invention program of Hunan province (2017DK2011).

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Hairpin-Contained i-Motif Based Fluorescent Ratiometric Probe for

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