Polymeric Optodes Based on Upconverting Nanorods for Fluorescent

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Polymeric Optodes Based on Upconverting Nanorods for Fluorescent Measurements of pH and Metal Ions in Blood Samples Liangxia Xie, Yu Qin,* and Hong-Yuan Chen State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, China S Supporting Information *

ABSTRACT: Optical thin films incorporating NaYF4:Er,Yb upconverting nanorods and chromoionophore ETH 5418 in hydrophobic polymer matrixes have been developed for the first time to measure pH and metal ions based on the ion-exchange mechanism. The absorption spectra of protonated and unprotonated ETH 5418 overlap the two emission peaks of upconverting material, respectively, which makes the inert nanorods ion-sensitive. Optodes for pH and metal ions (Na+, K+, Ca2+, and Cu2+) were investigated and exhibited excellent sensitivity, selectivity, and reproducibility. Because of excitation by the 980 nm laser source, detection in the near-infrared region at 656 nm, and high quantum yield of the nanorods in hydrophobic membrane, the proposed sensors have been successfully used in whole blood measurements with minimized background absorption and sample autofluorescence.

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sample to sample. Ion optodes can use absorption mode or fluorescent mode. Because of its high sensitivity, fluorescence measurement is more favorable in biological analysis. However, the most commonly used chromoionophores for bulk optodes to date are ETH-series including ETH 5294, ETH 2439, ETH 5418, ETH 5315, ETH 2412, ETH 7075, and ETH 7061, which are weakly or nonfluorescent. Meanwhile, the strong background absorption, autofluorescence, and scattering observed from biological samples such as whole blood in the UV and visible region significantly limit the application of traditional chromoionophores. Although blood fluorescence can be suppressed with optical isolation by covering the sensors with carbon black,11,12 developing sensors for direct measurements in whole blood and other biological samples are of great importance. Spectrofluorimetry in the near-infrared (NIR) region (650− 1000 nm) is an area of increasing interest. NIR light penetrates skin and overlaying tissue as deep as a few millimeters, and most biological matter exhibits relatively low levels of background interference in this region.13 In addition, biological samples such as whole blood shows weak absorption in the NIR region, thus long wavelength optical sensors are highly desirable.14 Among numerous fluorescent dyes, upconverting nanomaterials exhibit unique properties, such as the emission at visible and NIR wavelengths when excited with a 980 nm laser source. The large antistokes shift allows easy separation of the discrete emission peaks from the excitation source.15 The background absorption and sample autofluorescence are greatly reduced that leads to highly sensitive optical measurement. Unlike conventional organic dyes or quantum dots, upconvert-

evelopment of highly sensitive and selective sensors for ion detection in environmental and biological samples has gained tremendous attention in recent years. Measuring ion concentrations and imaging their spatial distribution in biological systems is of great interest for understanding physiological responses, such as signal transduction. Fluorescent sensors for ionic analytes have consistently demonstrated their potential in biomedical and environmental applications. For example, fluoro-ionophores for alkaline and alkaline-earth metal ions have been developed and commercialized for blood analysis. Such compounds containing recognition and transduction subunits in one molecule are selective and pH insensitive.1−3 However, the design and synthesis of fluoroionophores with desired selectivity and measuring range are usually challenging, and there are a limited number of ions for which indicator dyes are available. Ionophore-based ionselective optical sensors (bulk optodes) are a very versatile class of optical sensors and can reliably measure different ions under physiological or environmental conditions.4−10 Utilizing selective carriers originally developed for use in ion-selective electrodes (ISEs) optodes often possess much greater selectivity than optical sensors based on conventional spectrophotometric reagents. Ion optode membranes are composed of plasticized polymer, highly selective ionophore, ion exchanger, and a lipophilic pH indicator (chromoionophore) for signal transduction. Upon extraction of the target ion into the polymer phase, there is a concerted expulsion of a hydrogen ion from the chromoionophore into the aqueous phase in order to maintain electroneutrality within the polymer phase. This change in the degree of protonation of the chromoionophore can be monitored spectroscopically and used to quantify the activity of the primary ion. One drawback of such mechanism, however, is that sensors are obviously sensitive to pH in addition to the primary ion and measurements must be adjusted for changes in pH from © 2012 American Chemical Society

Received: November 11, 2011 Accepted: January 24, 2012 Published: February 9, 2012 1969

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using an external 1 W continuous-wave laser (980 nm) as the excitation source. Synthesis of NaYF4:Er,Yb nanorods. Nanorods NaYF4:Er,Yb were synthesized using a modified hydrothermal method according to the literature.23 In detail, NaOH (1.2 g, 30 mmol), water (7 mL), ethanol (12 mL), and oleic acid (22 mL) were mixed under agitation to form a homogeneous solution. Then 1.0 mmol (total amounts) of rare-earth chloride (2 mL, 0.5 M LnCl3, Ln: 78 mol % Y + 20 mol % Yb + 2 mol % Er) aqueous solution was added under magnetic stirring. Subsequently, 1.0 M aqueous NaF (5 mL) solution was added dropwisely to the above solution. The mixture was stirred for about 10 min, then transferred to a 50 mL autoclave, sealed, and hydrothermally treated at 195 °C for 16 h. The system was left to cool to room-temperature. Cyclohexane was used to dissolve and collect the products. The products were subsequently deposited by adding ethanol to the cyclohexane solution. The resulting mixture was then centrifuged to obtain powder samples. To obtain pure powders, the samples were washed with ethanol and water several times to remove oleic acid, sodium oleate, and other remnants. Preparation and Characterization of Optodes. For pH sensing, the sensing cocktail was prepared by dissolving the chromoionophore ETH 5418 (5 mmol/kg), cation-exchanger NaTFPB (5 mmol/kg), up-converting nanorods (2 mg), together with PVC and the plasticizer DOS or NPOE (1:2 by weight) to give a total cocktail mass of 100 mg, in 1 mL of THF. After vigorously shaking for 0.5 h, the mixture was under sonication for another 0.5 h. To obtain the optode thin film, a 50 μL aliquot of the sensing cocktail was deposited with a pipet onto quartz slides (35 mm ×11 mm) and remaining solvent was left to evaporate in a draft hood for at least 0.5 h prior to measurements. For metal ion sensing, ETH 5418 (5 mmol/kg), NaTFPB (10 mmol/kg), and ionophore (35 mmol/kg for Ca2+, 20 mmol/kg for others) were used in the cocktail solution. In all the measurements, each film-coated quartz slide was inserted into the custom-built quartz cuvette for detection. Ion activities are calculated from concentration and activity coefficients by the Debye−Hückel approximation that calibrates the ionic strength. All experimental data are the average of at least three measurements. Selectivity coefficients were determined by separate solution method (SSM) and calculated from the distance between the calibration curves for chosen deprotonation degree α value (α = 0.5) of ETH 5418.

ing nanoparticles exhibit neither photoblinking on the millisecond and second time scales nor photobleaching even with hours of continuous excitation,16 and their cytotoxicity is very low;17 thus, they have been successfully used in small animal imaging and even cell imaging.17,18 NaYF4 has been shown to be the most efficient host material for upconverting nanocrystals19,20 among various materials. The upconverting nanocrystals have been used in biosensing as labels to render a molecule luminescent or to report interactions over short distances (e.g., as a resonance energy transfer donor).21 With proper adjustment of surface chemistry, they become water-soluble and can be used for sensing bimolecules such as DNA,22−24 proteins,25 and enzyme activities21,26 in solution. However, the homogeneous sensing and detection approaches usually do not allow continuous and reversible measurements. Chemical sensors based on upconverting nanomaterials, on the other hand, can achieve these purposes by combining the nanomaterials with suitable recognition indicators. Wolfbeis and co-workers have developed several chemical sensors of this type, in sensing pH,27 ammonia,28 and carbon dioxide,29 respectively. The later two were performed with gas-permeable but proton-impermeable polymer, thus the pH change induced in the polymer matrix by the acidic or basic gases would be sensed. Recently, they also reported an oxygen sensor based on upconverting nanomaterials.30 In this work, we presented for the first time the optodes incorporating upconverting nanorods and chromoionophore in plasticized poly(vinyl chloride) matrixes for pH and metal ion sensing based on the combination of the traditional ionexchange mechanism and an inner filter effect. With the unique optical properties of the nanomaterial, the highly sensitive and selective measurements in the whole blood sample can be achieved.

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EXPERIMENTAL SECTION Materials. Oleic acid, YbCl3, ErCl3, and YCl3·6H2O were purchased from Alfa Aesar. NaF was obtained from Acros Organics. Tetrahydrofuran (THF), poly(vinyl chloride)high molecular weight (PVC), and bis(2-ethylhexyl) sebacate (DOS) were purchased from Sigma-Aldrich (Switzerland). Ca2+ selective ionophore AU-1 and Cu2+ selective ionophore N,N,N′,N′-tetracyclohexyl-3-thiaglutaric diamide were synthesized according to the literature,31,32 respectively. All the other ionophores, chromoionophores and all salts were obtained from Fluka (Switzerland). Cation-exchanger sodium tetrakis[3,5-bis(trifluoromethyl phenyl) borate (NaTFPB) was purchased from Dojindo Laboratories (Japan). Whole blood was freshly collected in heparin anticoagulated tubes from mice. Buffers. Phosphate buffers solutions (PBS) were prepared from 10 mM monobasic potassium phosphate, adjusting with 10 mM KOH solution. Gly-HCl buffer solution was prepared from 10 mM glycine adjusting with 0.1 M HCl solution to get pH 4.8. Diluted blood samples were prepared by diluting whole blood 20-fold in volume with required buffer solutions and used immediately. Water used for the experiments was double distilled and further purified with a Milli-Q filtration system. Instrumentation. X-ray diffraction (XRD) measurements were made with a Shimadzu XRD-6000 diffractometer using Cu Kα radiation (λ = 0.154 18 nm). UV−vis analyses were made with a Nonodrop-2000C spectrophotometer. Sizes and morphologies of nanorods were studied on a Hitachi S-4800 scanning electron microscope. Up-conversion fluorescence spectra were recorded on a ZolixScan ZLX-UPL spectrometer

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RESULTS AND DISCUSSION Synthesis and Characterization of NaYF4:Er,Yb. The upconverting nanorods used in this work were synthesized according to the literature.23 SEM images show that the nanorods are size uniform, with a diameter of 200 nm and length of 1 μm (Figure 1 inset). XRD pattern of the sample shows well-defined peaks. The peak positions and intensities from the experimental XRD pattern match closely with the calculated pattern for hexagonal NaYF4:Er,Yb (JCPDS no. 281192, see Figure S1 in the Supporting Information). The presence of the oleic acid ligand on the nanorods surface was confirmed via FT-IR. With modification by a highly lipophilic capping ligand, the nanorods could be easily dispersed in organic solvents. Upon excitation with a 980 nm laser, the nanorods display narrow green and red emission peaks centered at 542 and 656 nm, respectively, as shown in Figure 1. Among different morphology of the upconverting materials, the nanorods exhibit a high chemical stability and high quantum 1970

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but proton-impermeable polystyrene.28,29 In this work, upconverting nanorods together with cation-exchanger NaTFPB, chromoionophore ETH 5418 were incorporated in PVC-DOS matrix to produce a hydrophobic ion-exchangeable membrane for the first time. The highly lipophilic oleic acid capping makes the nanorods easily soluble in plasticized membrane. With shaking and sonicating, the cocktail became homogeneous and was then spread onto quartz slides. After evaporation of the solvent, the formed film with thickness calculated to be ∼7 μm was transparent, with no visible aggregation in it. Upon 980 nm excitation, the intensity of emission peaks of the proposed membrane is pH dependent. Figure 2 shows that

Figure 1. Main: Absorption spectra of ETH 5418 in PVC-DOS membrane containing cation-exchanger NaTFPB in 0.01 M NaOH (a) and 0.01 M HCl (b) aqueous solution, respectively; and luminescence emission of the nanorods in PVC-DOS membrane excited with a 980 nm laser. Inset: Scanning electron microscopy image of upconverting nanorods.

yields.33,34 They are known to have more intense luminescence and upconversion efficiency than spherical nanoparticles, and their luminescence intensity is independent of pH in the range from 2 to 11,27 which was experimentally confirmed and shown in Figure S2 in the Supporting Information. Such unique properties of upconverting nanorods make them an excellent choice of labeling material; however, there are also constraints of their applications in the sensing area. pH Sensing with Upconverting Nanorod Based Optode. In this work we first fabricated an upconverting optical pH sensor based on the ion-exchange principle and inner filter effect with hydrophobic PVC polymer matrix. The pH probe used here was a Nile Blue derivative, 9-dimethylamino-5-[4-(15-butyl-1,13-dioxo-2,14-dioxanonadecyl)phenylimino]benzo[a]phenoxazine (ETH 5418). It covers a wide pH response range from 4 to 12,35 and the pKa values were reported to be 8.56 in PVC-DOS and 11.72 in the PVCNPOE matrix.36 ETH 5418 has been used not only in pH sensing35 but also in metal ion detection.4,37,38 Although the smaller pKa value makes ETH 5418 useful for transition metal ion optode measurement at relatively low pH, the optical property of ETH 5418 is more suitable for applying the inner filter effect on the upconverting nanorods than other ETH chromoionophores. As shown in Figure 1, its absorption spectra of the protonated form (curve b in Figure 1) at low pH overlaps the red emission of the upconverting nanorods, while that of the deprotonated form at high pH (curve a in Figure 1) overlaps the green emission of the nanorods, and the isosbestic point is at 577 nm where no fluorescent signal is observed. Depending on whether ETH 5418 is present in its deprotonated or protonated form, the dye is expected to exert a strong inner filter effect on either green or red emission of the nanorods, which makes the originally inert upconverting material pH sensitive. Polymeric film was utilized to host nanorods and sensing components to meet the requirements of repeatable and reversible measurements. Previous works have reported successfully dispersing upconverting nanomaterials in various kinds of polymer matrixes as polyurethane hydrogel,27 PDMS,39 and gas-permeable

Figure 2. Main: Upconversion luminescence spectra of the sensor film containing ETH 5418 at pH values between 6 and 11 upon excitation of 980 nm. Inset: Upconversion luminescence intensity ratio of peak 656 nm to peak 542 nm of the sensing film containing ETH 5418 as a function of pH.

green emission at 542 nm decreases and red emission at 656 nm increases when the sample pH changes from 6 to 11. For each peak, the fluorescent response is linear but changing oppositely as the function of pH; therefore, ratiometric pH measurements with upconverting optodes can also be performed as shown in Figure 2 (inset). The results demonstrate that the intensities of emission at 542 nm decreases by about 27% at pH 11 and at 656 nm increase about 56% in comparison to those at pH 6. These behaviors were expected and can be explained by the inner filter effect between nanorods and ETH 5418. At low pH, the absorbance of ETH 5418 at 542 nm in the membrane is weak and the absorbance at 656 nm is strong. Thus the green luminescence emission (542 nm) of nanorods can be detected, while most of the red emission (656 nm) is absorbed by protonated ETH 5418. On increasing pH, fluorescence intensities increasing at 656 nm and decreasing at 542 nm are simultaneously detected because of the change of the protonation degree and absorption spectra of ETH 5418. At the same time, color of the membranes changing from light blue via purple to red can be observed visually. The repetitive cycling between pH 6 and 10 was performed with the same optode (see Figure 3), and the results show excellent reversibility and stability, indicating that no leaching of the components from the sensing membrane occurred during the measurements. Sun and Wolfbeis reported an upcoverting pH sensor utilized the inner filter effect between the pH indicator and upconverting material entrapped in the hydrogel matrix27 that uptakes the whole sample solution into the sensing membrane. In our work, the hydrophobic polymer film that would protect the dyes from direct contact with the sample 1971

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the very large amounts of hemoglobin (about 20 mM) in the red cells as well as the substantial scattering loss of the excitation and emission light in the blood matrix that contains components with relatively large dimensions.13 It is notable that the relatively strong fluorescent signal in whole blood measurements can hardly be obtained with traditional optical methods, not only because of the advantages of upconverting process and detection in the NIR region but also benefit from the entrapment of nanorods in hydrophobic membrane that reserves its high quantum yield. Metal Ion Sensing with Upconverting Optodes. One of the advantages of bulk optodes is the convenient generation of high selectivity toward a specific ion by using ionophores. Metal ion-selective optodes usually make use of the selective interaction of hydrogen ions with lipophilic pH indicators as chromoionophores, and by buffering the sample pH, the films can respond to cations under competitive ion exchange equilibrium.41 On the basis of this sensing principle, we further fabricated the metal ion optodes containing upconverting nanorods, ETH 5418, and various ionophores. The equilibrium between sample and membrane could be written as

Figure 3. Repetitive cycling of the pH sensor membrane between pH 6 and 10 by monitoring the luminescent intensity at 656 nm.

is permselective, while H+ and metal ions enter the membrane phase by the ion-exchange process. In addition, the application of ETH 5418 allows a ratiometric fluorescent measurement which is more favorable than single peak intensity detection. The upconverting nanorod-based pH sensor was further evaluated in blood samples. It is well-known that electrochemical techniques are much more routinely utilized for blood measurements of pH and electrolytes than optical methods due to the strong autofluorescence and absorption of the blood sample in the ultraviolet and visible light region.40 In this work, the direct fluorescent measurements were performed with an upconverting pH optode upon 980 nm excitation in whole blood. The green emission peak of nanorods at 542 nm is severely weakened, while 50% of the fluorescence at 656 nm is unaffected, so that the single peak change at 656 nm can be used for pH sensing in the blood sample. Whole blood itself is a well-buffered solution; therefore, we prepared the 20-fold diluted blood samples to obtain different pH values without a significant change in the background property. The upconversion luminescence spectra of membranes in different pH blood samples are shown in Figure 4. It is clearly seen that the peak

Iz +(aq) + n L(org) + zCH+(org) ⇌ ILnz +(org) + zC(org) + z H+(aq)

where Iz+ is the cation, L is the ionophore, and C is chromoionophore ETH 5418. When metal ions Iz+ come in contact with the optode, they are extracted into the film and concomitantly exchanged with hydrogen ions in order to conserve electroneutrality within the film, which results in the proton release from the optode and chromoionophore ETH 5418 changing from its protonated form to its deprotonated form. Therefore, because of the inner filter effect described above, increasing intensity at 656 nm and decreasing at 542 nm will be obtained. Sodium and calcium ion-selective optodes were prepared with sodium ionophore X and oxapentanediamide derivative AU-1. Considering the application in biological samples, measurements were first performed in tris-HCl buffer at pH 7.4. Upon excitation by a 980 nm laser, the luminescent intensity at 656 nm was monitored. However, the response range appeared in quite low ion concentration (data not shown) due to the relatively low pK a value of the chromoionophore ETH 5418, so a lower pH buffer glycineHCl (pH 4.8) was applied in the following work in order to shift the optode measuring range to higher values. As demonstrated in Figure 5, when measuring in aqueous solution, both the intensities at 656 nm and the intensity ratio of peak 656 nm to peak 542 nm increased along with the ion concentration increasing in the solutions. Titration curves obtained by single peak measurement and ratiometric method show the same response patterns. A sodium ion optode exhibited the response range between 10−5 and 10−2 M at pH 4.8. High Na+ concentration caused significant decrease of fluorescent intensity at both 542 and 656 nm; however, the ratio of the two peaks remained the same. Similar results were observed for calcium ion optodes, which shows the upper detection limit at 10−4 M. It is known that more free metal ions can be coextracted into the organic phase when ionophores are saturated in high concentration solution. The results suggested that free metal ions extracted into the membrane could change the luminescence property of the upconverting nanorods. Quenching of the luminescence of upconverting nanoparticles

Figure 4. Main: Upconversion luminescence spectra of the sensor membrane in diluted blood. Inset: Upconversion luminescence intensity of sensor membrane at 656 nm in buffer (●) and 20-folddiluted whole blood (▲), respectively, as a function of pH.

intensity at 656 nm increased linearly with the blood pH value increasing the same as in aqueous solutions, suggesting that the optode membrane functioned properly under more complex circumstances such as whole blood. Less strong emission at 656 nm compared to that in aqueous solution is mainly due to 1972

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Figure 5. Main: Response curves of upconverting nanorod-based (a) membrane with sodium X toward Na+ and (b) membrane with AU-1 toward Ca2+, by monitoring the luminescent intensity at 656 nm (i, iii), and the ratio of peak 656 nm to peak 542 nm (ii, iv). I0 is the intensity of the membrane in a pH 4.8 gly-HCl buffer solution only. Inset: Response curves of upconversion nanorods based Ca2+-selective membrane toward low Ca2+ concentrations by monitoring intensity at 656 nm (iii inset) and ratio of peak 656 nm to peak 542 nm (iv inset).

and NPOE (ε = 24), and the same measuring ranges and exchange constants have been observed, which indicated that nanorods decreases the polarity of PVC-NPOE matrix by their nonpolar capping ligands. Our results also suggested that oleic acid capped upconverting nanorods may have specific interactions with some metal ions although they are usually considered inert to the environment. Selectivity coefficients of the proposed optodes were determined by the separate solution method (SSM) and calculated from the distances between the calibration curves (shown in Figure S3 in the Supporting Information) for the chosen deprotonation degree α value (α = 0.5) of ETH 5418. Upconverting nanorod-based sensors show the same selectivity pattern as traditional ones. The Na+selective optode exhibited good selectivity toward Na+ over K+, Ca2+, and Mg2+. The selectivity coefficients were obtained as opt opt log KNa,K = −2.21 ± 0.15, log KNa,Ca = −5.69 ± 0.18, and log opt KNa,Mg = −6.67 ± 0.12. On the other hand, the calcium ion optode are less selective mainly because of the nature of the ionophore AU-1 and low polarity of the membrane. The opt opt selectivity coefficients were log KCa,K = −2.45 ± 0.08, log KCa,Na = opt −3.56 ± 0.10, and log KCa,Mg = −4.98 ± 0.04. Using valinomycin and N,N,N′,N′-tetracyclohexyl-3-thiaglutaric diamide as ionophores, we also prepared K+ and Cu2+ selective optodes. Both of them showed sensitive and selective detection of K+ and Cu2+ at pH 4.8 buffer solutions. The detection range is 10−7−10−4 M and 10−6−10−3 M, respectively (see Figure S4 in the Supporting Information).

by heavy metal ions in homogeneous solution has been reported,42 suggesting that formation of a ground-state and nonfluorescent complex between the upconverting nanoparticles and heavy metal ions might cause the decreasing in the quantum yield of the emission. However, the fluorescence decrease of nanorods in a nonpolar hydrophobic membrane caused by high concentration alkaline and alkaline-earth metal ions has not been reported before, which might be due to the influence of extracted metal ions on the capping ligands through electrostatic interaction. It is noticeable that the detection limits of Ca2+-selective optodes were much lower than those obtained with conventional chromoionophore ETH 5294 based optodes,31 in similar measuring conditions. The unusual behavior at low metal ionconcentration circumstances was particularly studied as shown in Figure 5 inset, and the curves have been normalized to the intensity at the maximum. It is clear that the Ca2+ sensing membrane can respond to even trace levels of the target ions, which cannot be solely explained by the lower pKa value of ETH 5418 (8.6) than ETH 5294 (11.4)36 in the PVC-DOS matrix. The upconverting nanorods might influence the properties of the sensing membranes. The long nonpolar alkyl chains capping on the surface of nanorods would change the polarity of the bulk phase. It is known that the optode response can rely on the polarity of the solvent due to the pKa value change of the chromoionophore. We prepared two upconverting optodes with different plasticizers DOS (ε = 3) 1973

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Because of the relatively low pKa value of ETH 5418, the optodes cannot measure the sodium ion concentration directly in undiluted whole blood at pH 7.4. We performed the sodium ion calibration of the sensor in diluted blood at pH 4.8 by the standard addition method and obtained similar results to these in aqueous solutions (data not shown). Further work on changing the response range with appropriate and more basic chromoionophores is in progress in our lab.

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CONCLUSIONS In summary, a metal ion and pH sensor based on the changes in the luminescence intensity of upconverting nanorods resulting from the absorption change of a pH sensitive chromoionophore ETH 5418 has been developed. The spectral overlap between the absorbance region of chromoionophore and the luminescence of the nanorods induces an inner filter effect depending on the pH or metal ion concentration in samples. Because of the excitation by the 980 nm laser source and emission in the NIR region, the measurements with proposed optical sensors eliminate the background absorption and autofluorescence. The immobilization of the upconverting nanorods in the lipophilic membrane phase further ensures the high fluorescent quantum yield. Such sensors exhibit high sensitivity and selectivity toward pH and different metal ions when applying for blood measurements. Similar to the traditional ion-selective optodes, the detection limit and dynamic range can be further tuned by varying the pH indicator with an appropriate pKa value for direct fluorescent measurements in biological samples.

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ASSOCIATED CONTENT

* Supporting Information S

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 86-25-83592562. Fax: 86-25-83592562. E-mail: [email protected].

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grants 21135002, 21075062, and 21121091).

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REFERENCES

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dx.doi.org/10.1021/ac203003w | Anal. Chem. 2012, 84, 1969−1974