Direct Fluorescent Detection of Blood Potassium by Ion-Selective

Aug 25, 2016 - The practical application was demonstrated by direct detection of K+ from real blood samples by two-photon fluorescence technology. To ...
8 downloads 13 Views 1MB Size
Download PDF
Article pubs.acs.org/ac

Direct Fluorescent Detection of Blood Potassium by Ion-Selective Formation of Intermolecular G‑Quadruplex and Ligand Binding Le Yang,† Zhihe Qing,*,‡ Changhui Liu,† Qiao Tang,† Jishan Li,† Sheng Yang,‡ Jing Zheng,† Ronghua Yang,*,†,‡ and Weihong Tan† †

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Molecular Science and Biomedicine Laboratory, Hunan University, Changsha 410082, P. R. China ‡ School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410004, P. R. China S Supporting Information *

ABSTRACT: G-quadruplex analogues have been widely used as molecular tools for detection of potassium ion (K+). However, interference from a higher concentration of sodium ion (Na+), enzymatic degradation of the oligonucleotide, and background absorption and fluorescence of blood samples have all limited the use of G-quadruplex for direct detection of K+ in blood samples. Here, we reported, for the first time, an intermolecular G-quadruplex-based assay capable of direct fluorescent detection of blood K+. Increased stringency of intermolecular G-quadruplex formation based on our screened G-rich oligonucleotide (5′-TGAGGGA GGGG-3′) provided the necessary selectivity for K+ against Na+ at physiological ion level. To increase long-term stability of oligonucleotide in blood, the screened oligonucleotide was modified with an inverted thymine nucleotide whose 3′-terminus was connected to the 3′-terminus of the upstream nucleotide, acting as a blocking group to greatly improve antinuclease stability. Lastly, to avoid interference from background absorption and autofluorescence of blood, a G-quadruplex-binding, two-photon-excited ligand, EBMVC-B, was synthesized and chosen as the fluorescence reporter. Thus, based on selective K+ ion-induced formation of intermolecular G-quadruplex and EBMVC-B binding, this approach could linearly respond to K+ from 0.5 to 10 mM, which matches quite well with the physiologically relevant concentration of blood K+. Moreover, the system was highly selective for K+ against other metal ions, including Na+, Ca2+, Mg2+, Zn2+ common in blood. The practical application was demonstrated by direct detection of K+ from real blood samples by two-photon fluorescence technology. To the best of our knowledge, this is the first attempt to exploit molecular G-quadruplex-based fluorescent sensing for direct assay of blood target. As such, we expect that it will promote the design and practical application of similar DNA-based sensors in complex real systems.

P

recognition elements.9 In the past few years, G-quadruplex analogues have proved to be potential recognition units for K+ via monovalent cation-induced formation of specific fourstranded helical conformation (G-quadruplex),10−13 and significant advances have been made in the development of selective biosensors for K+ based on G-quadruplex.14−18 However, to the best of our knowledge, despite the large number of studies, virtually all of them were limited to the detection of K+ in aqueous buffer systems, leaving the detection of K+ in real blood fluids to be elucidated. Three issues need to be addressed when using G-quadruplex analogues for the detection of blood K+. The first involves insufficient selectivity for K+ against competitive Na+ ions. K+ has a higher association constant than Na+ in G-quadruplex formation, but the typical physiological concentration of Na+ (∼145 mM) is much higher than that of K+ (∼5.0 mM) in the

otassium ion (K+), as an essential species in the human body, plays significant roles in regulating physiological functions, including heartbeat, muscular strength, nerve transmission, and renal function.1,2 In particular, the level of K+ in blood is a valuable indicator of health and clinical diagnosis, and direct effects of blood K+ on the cardiovascular system have been demonstrated. Normal potassium concentration in plasma ranges from 3.5 to 5.5 mM, and an increase or decrease of K+ in blood will result in hyper- or hypokalemia, subsequently causing an imbalance of blood pressure and cardiac rhythm. Especially, when its concentration is lower than 2.0 mM or higher than 7.0 mM, lethal complications will occur.3−5 In this context, the development of effective methods to detect blood K+ level is very necessary and significant. Toward this goal, various analytical techniques have been established, such as flame photometry,6 cold vapor technique with atomic absorption/emission spectrometry,7 and capillary electrophoresis.8 Apart from such conventional methods, electrodes, as an alternative, have been developed to detect blood K+ by using ion-selective chromoionophores as the © 2016 American Chemical Society

Received: July 13, 2016 Accepted: August 25, 2016 Published: August 25, 2016 9285

DOI: 10.1021/acs.analchem.6b02667 Anal. Chem. 2016, 88, 9285−9292

Article

Analytical Chemistry real bloodstream,17 and high concentrations of Na+ can also induce the formation of G-quadruplex.18,19 Thus, selective detection of K+ against higher concentration of competitive Na+ using traditional G-quadruplex is a challenge.18,20 Second, the response kinetics of G-quadruplex toward K+ may not be instantaneous.14,21 As such, the stability of the G-quadruplex structure in real blood samples presents another challenge as a result of enzymatic degradation of oligonucleotides in complex biological fluids.22−24 Finally, significant background absorption and strong autofluorescence from indigenous species in biological fluids, such as proteins, riboflavin, pyridoxines, and porphyrines, present barriers against the use of conventional absorption or fluorescence detection techniques.25−27 To address all of these issues, we herein proposed a Gquadruplex-based assay for direct fluorescent detection of K+ ions in physiological blood. Scheme 1 illustrates our proposed

EBMVC-B will be K+ concentration-dependent via K+-binding formation of intermolecular G-quadruplex. To the best of our knowledge, this is the first attempt to exploit G-quadruplex to design a fluorescent sensor for direct assay of blood target.

■

EXPERIMENTAL SECTION Chemicals and Materials. All oligonucleotides (Table S1) and the inverted thymine-modified oligonucleotide were purchased from SangonBiotech (Shanghai) Company, Ltd. The oligonucleotides were purified by high-performance liquid chromatography (HPLC). A stock solution of 100 μM for each oligonucleotide was prepared by dissolving them in sterilized deionized water and then diluting the samples to required concentrations before use. Calf serum, bovine serum albumin (BSA), and Dulbecco’s Modified Eagle’s medium (DMEM) were obtained from Sigma-Aldrich. Potassium chloride, sodium chloride, and other salts used in this work were at least analytical grade, commercially purchased from Dingguo Biotechnology Company, Ltd. (Beijing, China), and used without further treatment. The two-photon dye, ethyl-4-[3,6bis(1-methyl-4-vinylpyridium iodine)-9H-carbazol-9-yl)] butanoate (EBMVC-B), was synthesized from 3,6-dibromocarbazole, 4-bromobutyric acid ethyl ester, and 4-vinylpyridine, according to the method described in our previous work.33 The synthetic route for EBMVC-B is demonstrated in Figure S1, and the detailed process was the same as the previous work. Tris-HCl buffer (50 mM, pH 7.4) was prepared and used for the formation of G-quadruplex. Apparatus. All solutions were prepared using deionized water, which was obtained through a Millipore Milli-Q ultrapure water system (Billerica, MA). The pH values of corresponding solutions were measured by a model 868 pH meter (Orion). The circular dichroism (CD) spectra were collected on a MOS-500 spectropolarimeter (Biologic, France). Results of electrophoresis were determined by a ChemiDoc XRS+ imaging system (Bio-Rad). One-photon excitation (OPE) fluorescence measurements were performed on a PTI ASOC-10 Fluorescence System (Photo Technology International, Birmingham, NJ, U.S.A.). TPE fluorescence spectra were recorded on a DCS200PC single-photon counting (Beijing Zolix Instruments Co., Ltd.), which was equipped with a modelocked Ti: sapphire pulsed laser (Chameleon Ultra II, Coherent Inc.). Circular Dichroism Measurement. To demonstrate K+induced structural transformation of G-rich DNA, circular dichroism (CD) spectra of DNA were measured under different conditions. In brief, 2.5 μM full-length G-quadruplex DNA (G-quad, Table S1) or 5.0 μM G-rich oligonucleotide (oligo-3, Table S1) was, respectively, mixed with ions in 200 μL of Tris-HCl buffer and incubated for 4 h, followed by collection of CD spectra on a MOS-500 spectropolarimeter. The interval was set at 0.1 nm, and the spectral results were averaged from three scans ranging from 220 to 340 nm. The background interference of the buffer solution was deducted from the CD data. K+-Dependent Intermolecular G-Quadruplex DNA Screening. Based on the fluorescence enhancement of EBMVC-B via formation of intermolecular G-quadruplex, different G-rich sequences have been screened. Typically, 2.5 μM EBMVC-B were mixed into 500 μL of Tris-HCl buffer, and the resultant fluorescence was first recorded. Then, 500 nM Grich DNA were added into the above solution and incubated for 10 min, and the resultant fluorescence was measured again.

Scheme 1. Schematic Illustration for Highly Selective Detection of Blood Potassium Based on K+-Dependent Formation of Intermolecular G-quadruplex and Two-Photon Ligand Binding

approach. Different from previous G-quadruplex-based K+ sensors,12−18 G-quadruplex counterparts in this approach are driven by K+-dependent binding of two screened G-rich oligonucleotides (5′-TGAGGGAGGGG-3′) via intermolecular interaction. This increased stringency placed on intermolecular G-quadruplex formation may reduce target sensitivity, but the trade-off is the improved selectivity, especially worthwhile for a high concentration of blood K+ (∼5.0 mM). To increase longterm stability of oligonucleotides against enzymatic degradation, the screened sequence was modified with an inverted thymine nucleotide as a blocking group, whose 3′-terminus was connected to the 3′-terminus of the upstream nucleotide, improving the antinuclease stability of nucleic acid strands and enabling the application of nucleic acid probes in complex biological samples.28,29 Lastly, to avoid interference from biological absorption and autofluorescence, a two-photonexcited fluorophore, ethyl-4-[3,6-bis(1-methyl-4-vinylpyridium iodine)-9H-carbazol-9-yl)] butanoate (EBMVC-B), was designed and chosen as the binding ligand of G-quadruplex. It has been demonstrated that carbazole derivatives can interact with G-quadruplex analogues and that their fluorescence enhancements can be modulated by G-quadruplex.30−32 Moreover, with the significant two-photon excitation (TPE) displayed by EBMVC-B, it is expected that the fluorescence intensity of 9286

DOI: 10.1021/acs.analchem.6b02667 Anal. Chem. 2016, 88, 9285−9292

Article

Analytical Chemistry

Figure 1. Circular dichroism (CD) spectra of the full-length G-quadruplex analogue (G-quad, 5′-TGAGGGTGGGGAGGGTGGGGAA-3′) (A) and the screened subdouble oligonucleotide (oligo-3, 5′-TGAGGGAGGGG-3′) (B) in Tris-HCl buffer (50 mM, pH 7.4), responding to K+ and Na+. The concentrations of G-quad and oligo-3 are 2.5 and 5.0 μM, respectively.

Lastly, 5.0 mM K+ or 150 mM Na+ was introduced into the DNA/EBMVC-B solution and incubated for another 4 h, and the resultant fluorescence was also measured. From the fluorescence enhancement of EBMVC-B, the K+-dependent G-quadruplex DNA sequence could be successfully screened and used in further detection experiments. Electrophoresis Characterization. To demonstrate enzymatic cleavage of the nucleic acid probe in complex biological fluids, electrophoresis was applied to directly characterize the degradation of the nucleic acid probe. Typically, the unmodified oligo-3 probe (Table S1) and the inverted thymine nucleotide-modified oligo-6 probe (Table S1) of 3.0 μM were, respectively, added into a complex solution containing 50% calf serum and incubated for different time (0, 3, 5 h) at room temperature. Then, 12 μL of the resultant probe solutions was mixed with 3 μL of the nucleic acid dye SYBR Gold (100×) and 3 μL of loading buffer (6×). After they were stained for 5 min, electrophoresis was carried out by injecting 15 μL of the stained solution into the 4% agarose gel and running for 20 min at 80 V. Lastly, the electrophoresis results were imaged and recorded by a ChemiDoc XRS+ imaging system (Bio-Rad). Fluorescent Detection of K+ in Aqueous Solution. For detection of K+ in aqueous solution, different concentrations of K+ were added to 500 μL of Tris-HCl buffer containing 2.5 μM EBMVC-B and 1.5 μM oligo-3 (Table S1), followed by incubation for 4 h at room temperature. The OPE fluorescence spectra were collected from 500 to 700 nm with 450 nm excitation in a 0.2 × 1.0 cm2 quartz cuvette containing 500 μL of the measuring solution. To demonstrate the absence of interference from a physiological level of other ions, K+ detection was further carried out as described in the above procedure, except for prior addition of 150 mM Na+, 2.0 mM Mg2+ ions, 2.0 mM Ca2+ ions, and 5.0 μM Pb2+ into the buffer. To investigate the kinetics of the detection process, the realtime monitoring of fluorescence intensity was also implemented on the PTI ASOC-10 Fluorescence System. Direct Fluorescence Detection of K + in Blood Samples. To make a calibration curve of K+, an artificial blood buffer was used to prepare the standard solutions of K+.34 The artificial blood buffer contained 150 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, and 20 mg/mL bovine serum albumin in 50 mM Tris-HCl (pH 7.4) to mimic the complex electrolyte level, except for K+, in blood. Then, 2.5 μM EBMVC-B and 1.5 μM oligo-6 (Table S1) were mixed into 500 μL of artificial

blood buffer spiked with K+ of known concentrations. After a reaction time of 4 h, the two-photon fluorescence emission spectra were recorded in the range of 500 to 650 nm by a DCS200PC single-photon counting with a mode-locked Ti:sapphire pulsed laser. The output wavelength of the pulsed laser was centered at 810 nm with an average excitation power of 100 mW, a repetition rate of 80 MHz, and a duration time of 120 fs. The calibration curve was plotted between K + concentration and two-photon fluorescence intensity. For detection of blood K+, human blood samples were obtained from three volunteers, and four serum specimens were collected. Considering the linear range of detection and the probable K+ concentration, blood samples were diluted to 50% in volume with the artificial blood buffer before detection. Then, 2.5 μM EBMVC-B and 1.5 μM oligo-6 were introduced, and their two-photon fluorescence spectra were measured. The corresponding concentration of each blood sample was calculated though the calibration curve, which was compared with that obtained by coupled plasma mass spectrometry (ICPMS).

■

RESULTS AND DISCUSSION K -Selective Formation of Intermolecular G-Quadruplex. G-quadruplex DNAs have been screened as recognition units for K+,10−13 and various G-quadruplex-based sensors have been developed for K+ detection.14−18 However, high selectivity against Na+ is one of the challenges for direct detection of physiological potassium. Figure 1A shows the CD spectra of the full-length G-quadruplex DNA (G-quad, Table S1) induced by K+ or Na+ ions in Tris-HCl buffer (50 mM, pH 7.4). Typical ellipticity (θ) at 245 nm (negative) and 265 nm (positive) can be observed in the absence of any ion, indicating that G-quad can partially fold into parallel G-quadruplex structure in a spontaneous manner.13,20,35 After addition of 5.0 mM K+, both negative and positive peaks are obviously enhanced, suggesting that G-quad can be effectively recognized and stabilized to form intramolecular parallel G-quadruplex conformation by K+. However, with the addition of 150 mM Na+, the negative peak (245 nm) is weakened, while the positive peak (265 nm) is enhanced, which may result from topological transformation toward the hybrid quadruplex.19,35 Thus, when G-quad is used to directly detect physiological K+, it is necessarily affected by Na+. On the contrary, when our screened oligo-3 (Table S1) was applied as the recognition 9287

+

DOI: 10.1021/acs.analchem.6b02667 Anal. Chem. 2016, 88, 9285−9292

Article

Analytical Chemistry

(e.g., porphyrin and carbazole derivatives) can induce and stabilize G-quadruplex based on their ligand binding interaction.30−32,40 Although signal enhancement can be detected following the addition of monovalent cations (Na+ or K+), it is difficult to discriminate between K+ and Na+, leading to the failed construction of ion-dependent sensors. Therefore, to reduce interference arising from the direct effect of G-quad on EBMVC-B and achieve highly ion-selective signal generation, a series of subdouble G-quadruplex DNAs (oligo-1 to -5) were programmed to modulate EBMVC-B fluorescence (Figure S3). Compared with EBMVC-B itself, Figure 2B shows no obvious fluorescence change after mixing EBMVC-B with oligo-3, and no interference can be detected from the introduction of Na+ at high concentration (150 mM). On the contrary, significant fluorescence enhancement is detected with the addition of 5.0 mM K+. The fluorescence responses of EBMVC-B to K+ and Na+ induced by different DNA probes are summarized in Figure 2C by increasing signal-to-background ratio, (F − F0)/F0, where F0 is the fluorescence intensity of DNA/EBMVC-B, and F is that after the addition of K+ or Na+ ion. When oligo-3 is used as the recognition unit, almost no signal increase occurs with the addition of 150 mM Na+, whereas a much stronger increase in signal-to-background ratio is displayed for 5.0 mM K+ ((F − F0)/F0 = 2.26). In comparison to the other subdouble G-quadruplex DNAs, oligo3 displays greater selectivity of K+ against Na+, even at much higher concentration, this phenomenon may be due to the high sequence-dependence of the recognition between nucleic acid molecules and targets.41,42 To further illustrate DNA sequence specificity, the selectivity coefficient φ is introduced and defined as the following formula:43 φ = |[(F − F0)/F0]K+|/|[(F − F0)/F0]Na+|, where K+ and Na+ are at their physiological level (5.0 mM and 150 mM, respectively). From Figure 2D, a much larger selectivity coefficient of φ = 232 is displayed for K+ by oligo-3 compared to other sequences. Then, a binding constant of 1.27 × 103 M−1 is calculated for oligo-3 with K+ by the double reciprocal plot method (Figure S4),44 indicating a moderate binding between oligo-3 with K+. These collective results demonstrate that K+dependent modulation of EBMVC-B fluorescence can be effectively improved by the formation of intermolecular Gquadruplex and that oligo-3 is screened as the effective recognition element for K+ detection, even in the presence of high concentration of Na+, by using EBMVC-B as the signal reporter. These findings constitute the basis for construction of a direct fluorescence detection method toward blood K+, as proposed in this paper. Fluorescence Detection of K+ in Aqueous Solution. To get the best performance of K+ detection by using our proposed sensing system, the concentration of oligo-3 and two other important parameters, temperature and pH, which both affect the stability of the advanced structure of nucleic acid, were optimized. As shown in Figures S5−S7, 1.5 μM oligo-3, physiological pH (7.4), and ambient temperature (20 °C) were chosen as the optimal conditions and used in further detection experiments. Because of the promising proof-of-mechanism, under optimal conditions, the sensitivity of our proposed strategy for K+ detection was evaluated. First, in response to different concentrations of K+, fluorescence emission spectra of oligo3/EBMVC-B were recorded and are shown in Figure 3A. From the fluorimetric titration curves, the fluorescence emission intensity of EBMVC-B at 575 nm increases gradually as K+

element, only a characteristic positive CD peak at about 255 nm was observed in the absence of any ion (Figure 1B), indicating that oligo-3 itself maintains unfolded state in the buffer.36 After introduction of 5.0 mM K+, the positive peak (265 nm) is significantly enhanced, and the negative peak (245 nm) appears, confirming that K+ can effectively induce the formation of parallel intermolecular G-quadruplex structure of oligo-3 and that the change of CD spectra of oligo-3 is K+ concentration-dependent at the millimolar level (Figure S2), which matches quite well with the physiologically relevant concentration of blood K+. Interestingly, Na+ only exerts negligible influence on the CD spectrum of oligo-3, even at a much higher concentration (150 mM). Thus, these results verify that the strategy based on intermolecular G-quadruplex holds great potential for direct detection of blood potassium with sufficient selectivity against the physiological level of Na+. K+-Dependent Modulation of EBMVC-B Fluorescence via Intermolecular G-Quadruplex. The synthesized fluorescent dye EBMVC-B derived from carbazole and vinylpyridine includes cationic charges and a large flat aromatic structure, making it an excellent ligand complementary to Gquadruplex.37−39 Thus, EBMVC-B was used as the fluorescent ligand to monitor the formation of G-quadruplex, and its fluorescent properties upon interaction with different G-rich DNAs were investigated under the same conditions. Figure 2A shows weak fluorescence emission for EBMVC-B itself in TrisHCl buffer (50 mM, pH 7.4), while obvious fluorescence enhancement is observed following the addition of G-quad. This result is consistent with reports indicating that some dyes

Figure 2. One-photon fluorescence spectra of EBMVC-B (2.5 μM) under different conditions when G-quad (A) and oligo-3 (B) were used as the recognition elements, respectively; (C) Comparisons of increasing signal-to-background ratio ((F − F0)/F0) of EBMVC-B induced by 5.0 K+ or 150 mM Na+ by using different G-rich DNAs as the recognition elements; (D) Selectivity coefficient (φ) of each DNA toward K+, which is defined as |[(F− F0)/F0]K+/|[(F − F0)/F0]Na+|, where F0 is the fluorescence intensity of DNA/EBMVC-B, and F is that after the addition of the corresponding ion. All DNAs have a concentration of 500 nM, and the concentration of K+ and Na+ are 5.0 and 150 mM, respectively. The fluorescence was monitored at 575 nm with an excitation wavelength of 450 nm. 9288

DOI: 10.1021/acs.analchem.6b02667 Anal. Chem. 2016, 88, 9285−9292

Article

Analytical Chemistry

Figure 3. (A) Fluorescence emission spectra of the oligo-3/EBMVC-B (λex = 450 nm) with the addition of K+ in Tris-HCl buffer (50 mM, pH 7.4). The inset shows the response of oligo-3/EBMVC-B to different concentrations of Na+. (B) Fluorescence emission spectra and (C) signal change (F/ F0) of the oligo-3/EBMVC-B detection system toward different control ions. The concentrations for K+, Li+, Na+, Mg2+, Ca2+, and Pb2+ are 5.0, 2.0, 150, 2.0, 2.0, and 0.005 mM, respectively; NH4+, Cs+, Zn2+, Ni2+, and Cu2+ are used with a concentration of 0.02 mM. (D) Plot of the signal change (F/F0) vs ion concentration: (a) adding K+ only, (b) adding Na+ only, and (c) adding K+ in the presence of 150 mM Na+, 2.0 mM Li+, 2.0 mM Mg2+, 2.0 mM Ca2+, and 5.0 μM Pb2+. The inset shows the linear range of K+ detection by oligo-3/EBMVC-B, corresponding to curve b. The concentrations for EBMVC-B and oligo-3 used here are 2.5 μM and 1.5 μM, respectively.

Figure 4. (A) Real-time monitoring of oligo-3/EBMVC-B fluorescence emission as a function of adding different concentrations of K+. The concentrations for EBMVC-B and oligo-3 are fixed at 2.5 and 1.5 μM, respectively. (B) Electrophoresis characterization for the degradation of oligonucleotides incubated for various time in 50% calf serum. Compared to the naked probe (oligo-3), the ITN-protected probe (oligo-6) displays sufficient stability in complex biological fluid. The concentration of oligonucleotides for electrophoresis is 3 μM.

would be affected by other competitive species, different control ions of similar physiological level were detected by the oligo-3/EBMVC-B system. No interference on the EBMVC-B spectra resulted from ions common in blood, except K+ (Figure 3B), and a much higher signal-to-background ratio (F/F0) of 3.86 is displayed for 5.0 mM K+, whereas no signal enhancement is observed from other controlled ions of physiological level in comparison with the blank (Figure 3C). These results indicate that the detection system is highly K+selective. To further verify K+ specificity of the system, we carried out titration of K+ in the presence of potential

concentration increases from 0 to 30 mM. From the plots of fluorescence intensity versus ion concentration (curve a in Figure 3D), one can easily see that the signal generation of the oligo-3/EBMVC-B detection system is highly dependent on K+ concentration and that there is a linear detection range (R2 = 0.9749) from 0.5 to 10 mM with the lowest detectable concentration at 0.1 mM. Interestingly, adding Na+, instead of K+, in the detection system only has a negligible effect on the fluorescence signal when concentration is increased from 0 to 150 mM (inset in Figure 3A and curve b in Figure 3D). In addition, to test whether the performance for K+ detection 9289

DOI: 10.1021/acs.analchem.6b02667 Anal. Chem. 2016, 88, 9285−9292

Article

Analytical Chemistry

Figure 5. (A) Two-photon fluorescence intensity of oligo-6/EBMVC-B at 575 nm in Tris-HCl buffer (50 mM, pH 7.4) vs different excitation wavelength. (B) Two-photon fluorescence spectra of oligo-6/EBMVC-B with excitation of 810 nm in artificial blood buffer vs different K+ concentrations. The inset shows the linear relationship between the two-photon fluorescence enhancement (F/F0) of oligo-6/EBMVC-B against K+ concentration in artificial blood buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, and 20 mg/mL BSA, pH 7.4).

Fluorescent Detection of K+ in Blood Samples. The ubiquitous endogenous components in biological fluids can be concomitantly excited by short wavelengths; therefore, conventional OPE fluorescent measurement in complex biological fluids generally suffers from strong background fluorescence.25,26 Fortunately, TPE fluorophores with near-infrared excitation and visible emission have attracted high interest in the practical analysis of complex biological samples because they can efficiently overcome the limitations of OPE probes with lower self-absorption, lower biological autofluorescence and reduced photobleaching.34,45,46 Here, to evaluate the performance of the proposed strategy in complex conditions, we exploited the two-photon fluorescence property of EBMVC-B for K+ detection by virtue of its large flat conjugated system. Based on the relationship between two-photon fluorescence emission intensity of oligo6/EBMVC-B at 575 nm and near-infrared excitation wavelength in the absence and presence of K+ (Figure 5A), an optimal excitation wavelength of 810 nm was found, and a good signal-to-background ratio was displayed upon the introduction of K+ into the detection buffer. To further evaluate the performance of our proposed strategy for K+ detection in a complex environment, we first carried out OPE and TPE measurements of EBMVC-B in K+-containing complex biological fluids (cell culture media and human plasma), respectively. From the OPE fluorescence emission spectra (Figure S11A and Figure S12A), we can see a strong autofluorescence from the complex biological fluids, resulting in an insufficient signal response to the introduction of oligo-6/ EBMVC-B. As expected, TPE successfully depressed the endogenous autofluorescence from the complex media, and a significant fluorescence increase could be detected at 575 nm when oligo-6/EBMVC-B was added (Figure S11B and Figure S12B). These results indicate the practicality of direct detection of blood potassium based on the two-photon fluorescence response of oligo-6/EBMVC-B. Subsequently, for the calibration of K+ by two-photon fluorescence measurement, an artificial blood buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, and 20 mg/mL bovine serum albumin (BSA), pH 7.4) mimicking the complex electrolyte level in blood, except for K+, was prepared as the standard detection system.34 Aliquots of the mimetic solution were spiked with K+ of known concentrations, followed by the introduction of oligo-6/EBMVC-B. As shown in Figure 5B, the two-photon fluorescence intensity at 575 nm increases gradually as K+ concentration increases from 0 to 30

biologically interfering ions, which mimic blood electrolyte level, including 150 mM Na+, 2.0 mM Mg2+ ions, 2.0 mM Ca2+ ions and 5.0 μM Pb2+ ions (Figure S8). The result gave a titration curve almost superimposable with that obtained in the presence of K+ alone (curve c in Figure 3D), indicating sufficient sensitivity to meet the requirements for direct detection of blood K+. Stability of the Oligonucleotide in Complex Biological Fluids. Before application for direct detection of blood potassium, the reaction kinetics of K+ detection and the stability of oligo-3 in the biological environment were investigated. Upon the addition of different concentrations of K+, the real-time monitoring of fluorescence intensity of the oligo-3/EBMVC-B detection system was carried out (Figure 4A), and we see that several hours are required for reaching saturation of fluorescence signal. Unfortunately, in complex biological fluids, nucleic acid probes generally suffer from enzymatic degradation, especially during long-term incubation. Here, when the naked oligo-3 (lane 2, 4 and 6) is incubated in 50% calf serum for different time, electrophoretic characterization (Figure 4B) shows that it is gradually digested with the extension of incubation time. Meanwhile, the fluorescence intensity of the oligo-3/EBMVC-B system also decreases gradually (Figure S9A, C) after it is added into 50% calf serum. These results indicate the challenge of applying naked oligonucleotide in complex biological fluids. Therefore, to improve the stability of the oligonucleotide, oligo-3 was protected by modifying an inverted thymine nucleotide (ITN) at its 3′-end. This blocking group has been demonstrated to effectively resist enzymatic degradation.29,30 After modification, the oligo-3 sequence was renamed oligo-6. Upon using modified oligo-6 instead of oligo-3, no degradation is observed from the electrophoresis after incubation in 50% calf serum for different time (Figure 4B, lane 1, 3, 5), indicating that the integrity of ITN-modified oligo-6 is maintained in complex biological fluids. The effect of ITN on protecting nucleic acid strand from degradation is also verified through fluorescence signal (Figure S9B, C), a steady persistence of fluorescence is observed for oligo-6/EBMVC-B system over a longer period of time, indicating that oligo-6 holds good potential for application in complex biological fluids. K+ detection capability of the modified oligo-6 was then investigated, as well as naked oligo-3. Figure S10 shows that similar detection performance is demonstrated for oligo-3 and oligo-6, verifying that the ITN-modification does not weaken the capability for K+ detection. 9290

DOI: 10.1021/acs.analchem.6b02667 Anal. Chem. 2016, 88, 9285−9292

Analytical Chemistry

■

mM, and a good linear relationship (y = 0.1712x + 1.6996, R2 = 0.9905) between signal-to-background ratio (F/F0) and K+ concentration was obtained in the range of 0.5 to 10 mM (inset in Figure 5B). Finally, the direct two-photon fluorescent detection of K+ was carried out in real blood samples. Human blood samples were collected from a hypokalemic patient and two healthy volunteers, and their plasma was used after agglutination. Fifty percent of blood samples in the mimetic solution were detected, and their potassium concentration was calculated according to the linear calibration equation. Compared with the inductively coupled plasma mass spectrometry (ICP-MS), consistent values are determined by our proposed strategy with acceptable standard deviations (SD) (Table 1). These results successfully demonstrate the

ICP-MSa (mM)

sample 1e sample 2f sample 3g sample 3 + 4.0 mM

2.76 4.80 4.52 8.22

proposed method (meanb ± SDc) (mM)

recoveryd (%)

± ± ± ±

104.3 102.5 96.2 96.7

2.88 4.92 4.35 7.95

0.25 0.31 0.24 0.38

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b02667. Additional experimental results and figures as noted in text (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: +86-731-88822523. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

Table 1. Determination of Potassium Levels in Human Blood Samples by Our Proposed Method and Standard ICPMS sample

Article

ACKNOWLEDGMENTS We are grateful for financial support through the National Natural Science Foundation of China (21135001, 21405038, 21575018, 21505006, 21605008), the Foundation for Innovative Research Groups of NSFC (21521063), and the Hunan Provincial Natural Science Foundation (2016JJ3001).

■

a

ICP-MS experiments were conducted in the Analytical Center of the Research Institute of Mining and Metallurgy of Changsha. bMean of three determinations. cStandard deviation. dPercent recovery compared to ICP-MS. eThe sample was obtained from a hypokalemic volunteer. fThe sample was obtained from a healthy volunteer. gThe sample was obtained from another healthy volunteer.

REFERENCES

(1) Yu, S. P.; Canzoniero, L. M.; Choi, D. W. Curr. Opin. Cell Biol. 2001, 13, 405−411. (2) Kuo, H. C.; Cheng, C. F.; Clark, R. B.; Lin, J. J. C.; Lin, J. L. C.; Hoshijima, M.; Nguyêñ-Trân, V. T. B.; Gu, Y.; Ikeda, Y.; Chu, P. H.; Ross, J., Jr.; Giles, W. R.; Chien, K. R. Cell 2001, 107, 801−813. (3) Walz, W. Neurochem. Int. 2000, 36, 291−300. (4) Krishna, G. G.; Miller, E.; Kapoor, S. N. N. Engl. J. Med. 1989, 320, 1177−1182. (5) Schwartz, A. B. Angiology 1978, 29, 194−205. (6) Mayer, K. D.; Starkey, B. J. Clin. Chem. 1977, 23, 275−278. (7) Dipietro, E. S.; Bashor, M. M.; Stroud, P. E.; Smarr, B. J.; Burgess, B. J.; Turner, W. E.; Neese, J. W. Sci. Total Environ. 1988, 74, 249− 262. (8) Wan, Q. J.; Kubán, P.; Tanyanyiwa, J.; Rainelli, A.; Hauser, P. C. Anal. Chim. Acta 2004, 525, 11−16. (9) Seller, K.; Wang, K.; Bakker, E.; Morf, W. E.; Rusterbolz, B.; Spichiger, U. E.; Simon, W. Clin. Chem. 1991, 36, 1350−1355. (10) Weitzmann, M. N.; Woodford, K. J.; Usdin, K. J. Biol. Chem. 1996, 271, 20958−20964. (11) Walmsley, J. A.; Burnett, J. F. Biochemistry 1999, 38, 14063− 14068. (12) Lee, J.; Kim, H. J.; Kim, J. J. Am. Chem. Soc. 2008, 130, 5010− 5011. (13) Rehm, C.; Holder, I. T.; Groß, A.; Wojciechowski, F.; Urban, M.; Sinn, M.; Drescher, M.; Hartig, J. S. Chem. Sci. 2014, 5, 2809− 2818. (14) Ueyama, H.; Takagi, M.; Takenaka, S. J. Am. Chem. Soc. 2002, 124, 14286−14287. (15) He, F.; Tang, Y.; Wang, S.; Li, Y.; Zhu, D. J. Am. Chem. Soc. 2005, 127, 12343−12346. (16) Zhu, X.; Zhao, J.; Wu, Y.; Shen, Z.; Li, G. Anal. Chem. 2011, 83, 4085−4089. (17) Kim, B.; Jung, I. H.; Kang, M.; Shim, H. K.; Woo, H. Y. J. Am. Chem. Soc. 2012, 134, 3133−3138. (18) Olejko, L.; Cywinski, P. J.; Bald, I. Angew. Chem., Int. Ed. 2015, 54, 673−677. (19) Liu, L.; Shao, Y.; Peng, J.; Huang, C.; Liu, H.; Zhang, L. Anal. Chem. 2014, 86, 1622−1631. (20) Largy, E.; Marchand, A.; Amrane, S.; Gabelica, V.; Mergny, J. L. J. Am. Chem. Soc. 2016, 138, 2780−2792. (21) Qin, H.; Ren, J.; Wang, J.; Luedtke, N. W.; Wang, E. Anal. Chem. 2010, 82, 8356−8360.

practicality of our proposed approach for direct detection of blood potassium based on K+-dependent formation of intermolecular G-quadruplex and two-photon fluorescent ligand binding.

■

CONCLUSIONS In summary, we report here, for the first time, that Gquadruplex, as a recognition element, can be exploited for the direct fluorescent detection of blood potassium. The present approach has been engineered in unique ways that display obvious advantages and capabilities for K+ detection, which are not available in conventional G-quadruplex-based sensing systems. First, on the basis of the design of ion-selective formation of intermolecular G-quadruplex and ligand binding, a G-rich oligonucleotide has been successfully screened for the K+-dependent formation of intermolecular G-quadruplex. This provides excellent selectivity for K+ detection with no interference from competitive ions, even under physiological electrolyte conditions. Second, combined with antinuclease protection, the screened G-rich oligonucleotide exhibits improved stability, enabling its application in complex biological fluids. Finally, based on our synthesis and investigation of a two-photon fluorescent ligand, interference from background fluorescence and absorption of complex biological fluids was effectively avoided, and direct fluorescent detection of K+ was successfully verified in different blood samples, which is comparable to the standard ICP-MS approach. On the basis of these advantages, we expect that the design methodologies herein proposed can inspire new ideas for the engineering of reliable nucleic acid probes for a host of practical applications. 9291

DOI: 10.1021/acs.analchem.6b02667 Anal. Chem. 2016, 88, 9285−9292

Article

Analytical Chemistry (22) He, X.; Wang, K.; Tan, W.; Liu, B.; Lin, X.; He, C.; Li, D.; Huang, S.; Li, J. J. Am. Chem. Soc. 2003, 125, 7168−7169. (23) Komiyama, M.; Ye, S.; Liang, X.; Yamamoto, Y.; Tomita, T.; Zhou, J. M.; Aburatani, H. J. Am. Chem. Soc. 2003, 125, 3758−3762. (24) Shi, H.; He, X.; Cui, W.; Wang, K.; Deng, K.; Li, D.; Xu, F. Anal. Chim. Acta 2014, 812, 138−144. (25) Ma, C.; Bian, T.; Yang, S.; Liu, C.; Zhang, T.; Yang, J.; Li, Y.; Li, J.; Yang, R.; Tan, W. Anal. Chem. 2014, 86, 6508−6515. (26) Yi, M.; Yang, S.; Peng, Z.; Liu, C.; Li, J.; Zhong, W.; Yang, R.; Tan, W. Anal. Chem. 2014, 86, 3548−3554. (27) Yan, H.; He, L.; Ma, C.; Li, J.; Yang, J.; Yang, R.; Tan, W. Chem. Commun. 2014, 50, 8398−8401. (28) Shaw, J. P.; Kent, K.; Bird, J.; Fishback, J.; Froehler, B. Nucleic Acids Res. 1991, 19, 747−750. (29) Hicke, B. J.; Stephens, A. W.; Gould, T.; Chang, Y. F.; Lynott, C. K.; Heil, J.; Borkowsk, S.; Hilger, C. S.; Cook, G.; Warren, S.; Schmidt, P. G. J. Nucl. Med. 2006, 47, 668−678. (30) Chang, C. C.; Wu, J. Y.; Chien, C. W.; Wu, W. S.; Liu, H.; Kang, C. C.; Yu, L. J.; Chang, T. C. Anal. Chem. 2003, 75, 6177−6183. (31) Zhang, Y.; Wang, J.; Jia, P.; Yu, X.; Liu, H.; Liu, X.; Zhao, N.; Huang, B. Org. Biomol. Chem. 2010, 8, 4582−4588. (32) Chang, C. C.; Chien, C. W.; Lin, Y. H.; Kang, C. C.; Chang, T. C. Nucleic Acids Res. 2007, 35, 2846−2860. (33) Tang, Q.; Wang, N.; Zhou, F.; Deng, T.; Zhang, S.; Li, J.; Yang, R.; Zhong, W.; Tan, W. Chem. Commun. 2015, 51, 16810−16812. (34) Xie, L.; Qin, Y.; Chen, H. Y. Anal. Chem. 2013, 85, 2617−2622. (35) Monchaud, D.; Yang, P.; Lacroix, L.; Teulade-Fichou, M. P.; Mergny, J. L. Angew. Chem., Int. Ed. 2008, 47, 4858−4861. (36) Mohanty, J.; Barooah, N.; Dhamodharan, V.; Harikrishna, S.; Pradeepkumar, P. I.; Bhasikuttan, A. C. J. Am. Chem. Soc. 2013, 135, 367−376. (37) Wang, X.; Huang, J.; Zhou, Y.; Yan, S.; Weng, X.; Wu, X.; Deng, M.; Zhou, X. Angew. Chem., Int. Ed. 2010, 49, 5305−5309. (38) Yuan, L.; Tian, T.; Chen, Y.; Yan, S.; Xing, X.; Zhang, Z.; Zhai, Q.; Xu, L.; Wang, S.; Weng, X.; Yuan, B.; Feng, Y.; Zhou, X. Sci. Rep. 2013, 3, 1811. (39) Tian, T.; Song, Y.; Wang, J.; Fu, B.; He, Z.; Xu, X.; Li, A.; Zhou, X.; Wang, S.; Zhou, X. J. Am. Chem. Soc. 2016, 138, 955−961. (40) Fan, D.; Zhu, J.; Liu, Y.; Wang, E.; Dong, S. Nanoscale 2016, 8, 3834−3840. (41) Shangguan, D.; Li, Y.; Tang, Z.; Cao, Z. C.; Chen, H. W.; Mallikaratchy, P.; Sefah, K.; Yang, C. J.; Tan, W. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 11838−11843. (42) Torabi, S.; Wu, P.; McGhee, C. E.; Chen, L.; Hwang, K.; Zheng, N.; Cheng, J.; Lu, Y. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 5903− 5908. (43) Yang, R.; Jin, J.; Chen, Y.; Shao, N.; Kang, H.; Xiao, Z.; Tang, Z.; Wu, Y.; Zhu, Z.; Tan, W. J. Am. Chem. Soc. 2008, 130, 8351−8358. (44) Agarwal, S.; Jangir, D. K.; Mehrotra, R. J. Photochem. Photobiol., B 2013, 120, 177−182. (45) Mao, Z.; Hu, L.; Dong, X.; Zhong, C.; Liu, B. F.; Liu, Z. Anal. Chem. 2014, 86, 6548−6554. (46) Zeng, L.; Chen, S.; Xia, T.; Hu, W.; Li, C.; Liu, Z. Anal. Chem. 2015, 87, 3004−3010.

9292

DOI: 10.1021/acs.analchem.6b02667 Anal. Chem. 2016, 88, 9285−9292