Direct Fluorescent Measurement of Blood Potassium with Polymeric

Feb 15, 2013 - ABSTRACT: We proposed here a potassium selective optode incorporating. NaYF4:Er,Yb upconverting nanorods and chromoionophore ETH ...
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Direct Fluorescent Measurement of Blood Potassium with Polymeric Optical Sensors Based on Upconverting Nanomaterials 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: We proposed here a potassium selective optode incorporating NaYF4:Er,Yb upconverting nanorods and chromoionophore ETH 5294 together in hydrophobic polymer matrixes and the response of the optode is based on changes in the upconverting luminescence intensity induced from the absorption change of the proton sensitive chromoionophore. The optode was used for determination of the potassium content in the whole blood sample for the first time. Because the excitation source of 980 nm, as well as the emission wavelength, lies in the near-infrared region, the background absorption and autofluorescence of the biological sample could be eliminated, and ensure the sensitivity and selectivity of the sensor. The potassium levels of sheep plasma and whole blood samples obtained by the optode were comparable with the results obtained by ICPMS and ISE methods, providing possibilities for further application in clinical diagnosis.

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region. The background interference could be suppressed by the optical isolation technique.9 AVL Scientific Corporation introduced an OPTI system which was later acquired by Roche that could rapidly detect eight analytes in blood optically.10 The measurements of cationic electrolytes in the system are mainly based on the combination of fluoro-ionophores, incorporating both recognition and signal transduction subunits into one molecule11−14 and an additional optical isolation layer we mentioned above. The existence of isolation requires analytes to diffuse one more layer to reach the sensor, so developing optodes which are applicable in whole blood without any optical-dealing processes is still essential. Compared with fluoro-ionophores, the major disadvantage of bulk optodes is their cross-sensitivity to pH due to the usage of chromoionophore, so measurements must be adjusted for changes in pH from sample to sample. However, considering the blood is a self-buffered sample, such drawback is less problematic. In addition, the direct application of the fluoroionophores into a whole blood sample requires that both excitation and emission wavelength lie in the near-infrared (NIR) window to avoid background interference, which might bring in large conjugated groups. The dynamic range and selectivity of recognition subunits should also be taken into consideration, which makes the design and synthesis more challenging. On the other hand, bulk optodes have the key advantage of having a conveniently tunable response range through adjusting the amount of chromoionophore and ionophore, and introducing a second inert fluorescent probe into the bulk phase will combine both the advantages of the

otassium is one of the elements that is essential to the human body. Especially as a blood electrolyte, it has direct effects on the cardiovascular system.1 Changes of the extracellular potassium level in blood might cause hyper- or hypo-kalemia, which could have a dangerous effect on cardiac rhythm, and ultimately heart function, as well as on blood pressure regulation.2 Therefore, monitoring the potassium level in whole blood is very significant for clinical analysis. Initially, flame photometry was used for determining the blood electrolytes. Many other methods were developed afterward, including atomic absorption/emission spectrophotometry (AAS/AES), and capillary electrophoresis (CE).3 Today, the most popular method is ion-selective electrodes (ISEs), which have been widely used in commercial blood analyzers for rapid detection. Since most clinical analyses are spectrophotometric, when ISEs are built into blood analyzers, they often require a special compartment, special service, and additional calibration. In this situation, optical techniques would be more adaptable. As an alternative, polymeric bulk optodes have been used in detecting blood electrolytes.4 By introducing a second ionophore for a reference ion (often a lipophilic pH indicator called chromoionophore) into the sensor membrane, monitoring the change in the degree of protonation of the chromoionophore could quantify the activity of the primary ion since the response mechanism is based on competitive ion-exchange equilibrium.5 Because both ISEs and optodes depend on ionophore chemistry, their selectivity properties should be quite similar, and in practice, the optical and ISE systems should possess comparable selectivities. The application of bulk optodes in the detection of blood electrolytes is mainly in plasma or serum instead of whole blood samples,4,6−8 due to the strong background absorption, significant autofluorescence, and scattering observed from whole blood in the UV and visible © 2013 American Chemical Society

Received: December 20, 2012 Accepted: January 31, 2013 Published: February 15, 2013 2617

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probe and of the traditional bulk optodes.15,16 The second probe we used here is upconverting nanorods. Upconverting nanophosphors, typically excited with a NIR 980 nm laser source, are capable of converting low-energy light to higher-energy light through a multiphoton process, thus emitting light at the visible and NIR region, with a large antistokes shift that allows easy separation of the emission and excitation peak, therefore, significantly minimizing background autofluorescence, photobleaching, and photodamage to biological specimens.17 Because of such unique properties, the upconverting materials have been widely used as biomarkers in the biomatter imaging field,18−20 including vascular imaging.21,22 However, comparing with numerous work that making bioassays and performing homogeneous sensing based on the use of upconverting nanomaterial, applications for purposes of reversible optical chemical sensing are much less reported,23−27 especially when used in blood sample measurements. In our previous work, upconverting nanorod-based polymeric optodes were fabricated and used to determinate metal ion activity in buffer solutions at low pH (pH = 4.8), and also initially demonstrated that such sensors were promising for blood sensing.27 The low pH buffer was utilized due to the usage of chromoionophore ETH 5418, which has relatively low pKa.28 The early results suggested that by optimizing sensor composition, we could fabricate optodes meeting the demand of detecting electrolytes in whole blood samples at physiological pH. Therefore, we present here a modified K+ selective optode which incorporates upconverting nanorods and chromoionophore in plasticized poly(vinyl chloride) matrixes based on the combination of a traditional ion-exchange mechanism and inner filter effect. Owing to the NIR excitation nature of upconverting materials, the fabricated sensors were successfully used to detect potassium concentration in unknown sheep plasma and whole blood samples, with results comparable to that obtained by ISEs or ICPMS.

and whole blood were diluted 10-fold in volume with a 10 mM Tris-HCl buffer, and the pH was adjusted to 7.4. The concentrations of K+ in 10-fold-diluted sheep plasma and whole blood were determined by standard addition with spikes of buffered solutions with known potassium concentrations. Preparation of Sensor Membranes. The sensing cocktail was prepared by dissolving the chromoionophore ETH 5294 (5 mmol/kg), cation-exchanger NaTFPB (10 mmol/kg), K+ selective ionophore BME-44 (20 mmol/kg), upconverting nanorods (2 mg), together with PVC and the plasticizer DOS (1:2 by weight) to give a total cocktail mass of 100 mg in 1 mL of THF. The cocktail was vigorously shaken for 0.5 h and under sonication for another 0.5 h before use. To obtain the optode membrane, a 50 μL aliquot of the cocktail was deposited with a pipet onto quartz slides (35 mm × 11 mm) and the remaining solvent was left to evaporate in a draft hood for at least 0.5 h prior to measurements to get a sensing membrane with thickness calculated to be ∼7 μm. Methods. In all the measurements, each film-coated quartz slide was inserted into the custom-built quartz cuvette for detection. Ion activities in aqueous solutions are calculated from concentration and activity coefficients by Debye−Hückel approximation that calibrates the ionic strength. All experimental data are the average of at least three replicate measurements. Response time and reversibility tests were taken by switching the optode between 10−2 M and 10−4 M KCl solution and recording the intensity at 656 nm continuously for at least 10 min at each concentration. Selectivity coefficients were determined by the Separate Solution Method (SSM), and calculated from the distance between the calibration curves for chosen deprotonation degree α value (α = 0.5) of ETH 5294 at pH 7.4. The determinations of pKa of ETH 5294 and the binding constant measurement of BME-44 in the upconverting nanorods-based PVC−DOS matrix were performed using a segmented sandwich-membrane method according to the respective literature (see the Supporting Information)28,29. Instrumentation. The phase and structural purity of upconverting nanorods was confirmed with X-ray diffraction using a Shimadzu XRD-6000 diffractometer. The morphology was studied on a Hitachi S-4800 scanning electron microscope. Upconverting fluorescence spectra were recorded on a ZolixScan ZLX-UPL spectrometer using an external 1 W continuous-wave laser (980 nm) as the excitation source.

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EXPERIMENTAL SECTION Materials. Oleic acid, YbCl3, ErCl3, and YCl3·6H2O were obtained from Alfa Aesar. NaF was purchased from Acros Organics. Tetrahydrofuran (THF), poly(vinyl chloride) high molecular weight (PVC), bis(2-ethylhexyl) sebacate (DOS), and all salts were purchased from Sigma-Aldrich (Switzerland). Cation-exchanger sodium tetrakis-[3,5-bis(trifluoromethyl phenyl) borate (NaTFPB) was purchased from Dojindo Laboratories (Japan). The chromoionophore 9-(diethylamino)-5-octadecanoylimino-5H-benzo[a]phenoxazine (ETH 5294), valinomycin, and potassium ionophore III, 2-dodecyl2-methyl-1,3-propanediyl bis[N-[5′nitro-(benzo-15-crown-5)4′-yl]carbamate] (BME-44) were obtained from Fluka (Switzerland). Sheep plasma and whole blood were both purchased from Nanjing Bianzhen Biotechnology (China). Synthesis of NaYF4:Er,Yb Nanorods. Nanorods NaYF4:Er,Yb [1 μm (L.) × 200 nm (d.m.)] were synthesized using a hydrothermal method (see the Supporting Information), according to our previous work,27 and used as-prepared. Sample Preparation. All samples were prepared with 10 mM Tris, and the pH was adjusted with 0.1 M HCl to 7.4. Artificial blood solutions (ABS) mimicking the electrolyte level except for K+ in whole blood contained 140 mM NaCl, 1 mM MgCl2, and 2 mM CaCl2 in 10 mM Tris-HCl buffer at physiological pH.8 This recipe was used for the preparation of standard solutions for the calibration of K+. The sheep plasma

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RESULTS AND DISCUSSION 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, which could be written as Iz +(aq) + n L(org) + zCH+(org) ⇌ ILnz +(org) + zC(org) + z H+(aq)

(1)

eq Iz+ is the analyte cation; L is the ionophore, and C is the chromoionophore. 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 2618

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ETH 5294 and the binding constant of BME-44 in the upconverting nanorod-based PVC−DOS matrix were studied. The obtained pKa and binding constant values are 11.74 and 7.52, respectively, which are comparable to the literature values of 11.41 and 7.84.28,29 The results confirm that adding upconverting nanorods induces negligible influence to the membrane matrix, as long as the sensor functions under ionexchange equilibrium. K+ Selective Upconverting Nanorod-Based Optode Performance in Buffers. As demonstrated in Figure 2, when

from the optode and the chromoionophore changing from its protonated form to its deprotonated form. For a specific analyte ion, the dynamic range of the bulk optodes can be tuned by adjusting the pH of the sample, ion carriers with different binding constants, or the chromoionophore with different basicity. The widely used chromoionophore ETH 5294 (see Figure S1 of the Supporting Information for the structure) was chosen here. The sensors were designed based on an ion-exchange mechanism, and the inner filter effect between nanorods and ETH 5294. As shown in Figure 1, the absorption spectra of the

Figure 1. Absorption spectra of ETH 5294 in the PVC−DOS membrane containing the cation-exchanger NaTFPB in (a) 0.01 M NaOH and (b) 0.01 M HCl aqueous solution, respectively. Luminescence emission of the nanorods in PVC−DOS membrane excited with a 980 nm laser.

protonated form of ETH 5294 overlaps the red emission of the upconverting nanorods, while that of the deprotonated form overlaps the green emission of the nanorods. According to the ion-exchange mechanism we described above, the concentration change of the target ion (in this work, K+) could result in the deprotonation degree change of the chromoionophore. With dependence on whether ETH 5294 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, so K+ concentration can be determined. The K+ selective ionophore valinomycin was used first. However, due to the strong binding affinity of the ionophore to the target ion, the upper detection limit was around 10−4 M (data not shown), which is almost 10-fold below the physiological K+ concentration in blood, so we changed the ionophore to BME-44, which has a lower reported binding constant to K+ (7.84) than valinomycin (10.10) in the PVC− DOS matrix.29 Since the response of ion-selective optodes proposed here are based on competitive ion-exchange equilibrium, it is necessary to demonstrate that the sensing components function properly at the presence of upconverting nanorods. We were concerned that the long alkyl chains of the capping ligand oleic acid on the surface of the nanorods might change the bulk phase property. We were also concerned that the possible existence of free carboxyl groups might influence the apparent ionophore binding constant due to the chance of the partly ligand-detachment when the proton is extracted into the bulk phase during the optode protonation process.30 So, the pKa of

Figure 2. (a) Upconversion luminescence spectra of the upconverting nanorod-based membrane with BME-44 toward potassium from 10−5 to 10−2 M at pH 7.4 upon excitation of 980 nm. (b) Response curve of the K+ selective membrane by the intensity ratio of peak 656 nm to peak 542 nm. (c) Response curve of the K+ selective membrane by monitoring the luminescent intensity at 656 nm. I0 is the intensity of the membrane in a 10 mM pH 7.4 Tris-HCl buffer solution only. R2 represents the correlation coefficients of linear fitting of each curve within the concentration range from 10−4 to 10−2 M.

measurements were taken in Tris-HCl (pH 7.4) buffered KCl solutions, both the intensities at 656 nm and the intensity ratio of peak 656 nm to peak 542 nm increased along with the increased ion concentration. Titration curves obtained by single peak measurement and the ratiometric method show the same response patterns. The response range is from 10−4 M to 10−2 M, which covers the physiological level of K+ in blood, and the correlation coefficients, R2, of the linear fitting within this range are also shown in Figure 2. For the calibration (with log Kex = −4.45 for K+) at half-protonation of the chromoionophore (α = 0.5), the corresponding K+ activity is around 0.7 mM, which is close to the range of K+ activity in a 10-fold-diluted blood sample. High K+ concentration caused a simultaneous decrease of fluorescent intensity at both 542 and 656 nm, similar to what we observed before;27 however, the ratio of the two peaks remained uninfluenced. The decrease only happened when the K+ concentration was as high as 10−1 M, which was not within the response linear range (see Figure S3 of the Supporting Information). 2619

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selectivity coefficients calculated according to the literature31 based on the average electrolyte levels in blood.32 The results demonstrate that the sensor has sufficient selectivity, considering utilization in blood samples. Because of the abundant concentrations of common interferences for K+, it is necessary to calibrate the sensor in a fixed background of interferences using artificial blood solutions (ABS), which mimic the electrolyte level in whole blood (pH 7.4). The upconverting nanorod-based K+-selective optodes were calibrated in such solutions, and the calibration curves are shown in Figure 4, using the fluorescence intensity ratio of peak 656 nm to peak 542 nm and peak 656 nm itself. It is more clearly seen using the protonation degree of ETH 5294 (1-α) (see Figure S4 of the Supporting Information) that analyte concentrations below 10−4 M are biased from the theoretic curve (with log Kex = −4.30 for K+). However, from 10−4 to 10−2 M is still the linear response range. Such a satisfactory response range illustrates that the optodes are capable of maintaining sufficient selectivity in a background of blood electrolyte level and are suitable for physiological measurements. The correlation coefficient, R2, of the linear fitting within the range from 10−4 to 10−2 M is also shown in Figure 4. All detections were carried out under a 1 W 980 nm laser as the external excitation source. However, water has been reported to have a huge absorption peak around 980 nm,33 and an overheating effect under continuous irradiation at this wavelength has been observed.34 Since temperature might influence the selectivity of certain kinds of ionophores in the PVC-based matrix used as electrodes,35 and the upconverting luminescence is also found to be temperature dependent,36 temperature changing in the sample solution should be considered here. We placed 3 mL of aqueous solution in a custom-built quartz cuvette and put it under a 1 W excitation source with a starting temperature at 24 °C. In the initial 5 min, the elevation rate was approximately 1 °C/min (see Figure S5 of the Supporting Information), which seemed to be relatively quick. However, the spectra curve for each concentration was obtained within 1.5 min, during which period the temperature changed no more than 2 °C. In addition, the response curve in Figure 3 also shows that the luminescence intensity upon the 980 nm excitation at a certain concentration is constant over time. The results indicate that the heating effect was negligible

The optodes exhibited good reproducibility when doing the repetitive cycling between 10−2 and 10−4 M KCl solutions by recording the intensity at 656 nm continuously (Figure 3). The

Figure 3. Repetitive cycling of the K+ selective optode between 10 mM Tris-HCl buffered 10−2 and 10−4 M KCl solution at pH 7.4 by monitoring the luminescent intensity at 656 nm.

response time is within 10 min, so measurements were performed after a 10 min incubation in each sample concentration to allow the optodes to reach the equilibrium condition. Selectivity coefficients were determined by Separate Solution Method (SSM) and are listed in Table 1, as well as the required Table 1. Observed Exchange Constants and Selectivity Coefficients and Required Selectivity Factors for Upconverting Nanorod-Based Potassium-Selective Optodea ion

log Kex

log Kopt ij

log Kopt ij (required for 10-fold diluted blood)

log Kopt ij (required for undiluted blood)

K Na Ca Mg

−4.45 −7.70 −18.5 −18.0

− −3.25 −6.23 −5.73

− −3.14 −1.39 −0.92

− −3.09 −1.31 −0.88

a

These were in buffered solutions at pH 7.4. The required log Kopt ij values were calculated according to ref 31 using the Separate Solutions Method (SSM) and assuming a 2% error in the sensor response. The ion activity data used for the calculation were obtained from ref 32.

Figure 4. Response curve of K+ selective membrane with a constant background of interfering ions in the artificial blood solution (ABS) mimicking the electrolyte level in undiluted whole blood. (a) Using the intensity ratio of peak 656 nm to peak 542 nm (b) by monitoring the luminescent intensity at 656 nm. I0 is the intensity of the membrane in a 10 mM pH 7.4 Tris-HCl buffer solution only. R2 represents the correlation coefficients of linear fitting of each curve within the concentration range from 10−4 to 10−2 M. 2620

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Figure 5. (a) Upconverting luminescence spectra and (b) data points and a calculated calibration curve of the K+-selective optodes in 10-fold-diluted whole blood for the standard addition, with spikes of known potassium concentrations.

in the measurements we performed here, but careful temperature control is needed in future experiments. K+-Selective Upconverting Nanorod-Based Optode for Blood Measurements. The goal of this study was to develop upconverting nanorod-based optodes for potassium determination in blood samples. For the calibration in ABS solution mimicking the electrolyte level in whole blood, at halfprotonation of the chromoionophore (α = 0.5), the corresponding K+ activity is around 0.5 mM. In consideration of the normal K+ level in blood, as well as the linear range and sensitivity of the proposed optodes, sheep plasma and whole blood were 10-fold diluted in volume at physiological pH, followed by standard addition with the K+ solution of known concentration at the same pH. As we demonstrated earlier, both the fluorescent intensity ratio of peak 656 nm to peak 542 nm and the peak 656 nm intensity itself have a linear response range from 10−4 to 10−2 M. For 10-fold-diluted plasma, peak ratios were used to determine the K+ concentration. However, for 10-fold-diluted whole blood, because the green emission of the nanorods was severely weakened due to the background absorption in the blood matrix, the intensity of peak 656 nm was chosen. Aliquots of buffer solutions with known potassium concentrations were spiked separately into the sample, and after each spike, a response spectra curve was obtained, as shown in Figure 5a. The potassium values were calculated by a linear least-squares regression function of the luminescence intensity versus log aK+. An example of the linear fitting response curve, and also the data points, are shown in Figure 5b. Each sample was measured at least three times. The acquired data were summarized in Table 2. Clearly, the plasma results obtained by ICPMS, ISEs, and upconverting nanorod-based optodes are consistent with each other. Notably, the obtained potassium concentration in sheep

whole blood is much higher than the normal level. It might be due to partial hemolysis of red blood cells in the whole blood we purchased; that leaking of intracellular potassium may have caused an elevation of an extracellular potassium level. Overall, this work has demonstrated that upconverting nanorod-based K+-selective optodes could function properly and accomplish accurate detection in complex circumstances as whole blood, although it is commonly known that electrochemical techniques are much more routinely utilized for blood measurements of electrolytes than are optical methods, due to the strong autofluorescence and absorption of blood samples in the ultraviolet and visible light region; we illustrated here that the two methods are comparable. To minimize the inconsistency induced by hemolysis, our measurements by both methods (ISEs and optodes) were performed during the same day. The results obtained are in agreement with each other. The recoveries are 99.7% for plasma (using ICPMS results as the standard) and 108.0% for whole blood, with a relative standard deviation (RSD) of less than 5%. The above results have exhibited the applicability of this upconversion-based optode in a real sample for electrolyte quantification.

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CONCLUSIONS In summary, we proposed for the first time a potassiumselective optode based on changes in the upconverting luminescence intensity induced from the absorption change of the proton sensitive chromoionophore ETH 5294 for the direct fluorescent measurement of potassium ions in physiological blood samples. Because the excitation source of 980 nm and the emission wavelength are both in the near-infrared region, the background absorption and autofluorescence of the biological sample could be maximally eliminated, which ensures the sensitivity and selectivity when utilizing the upconverting nanorod-based optodes for blood measurements. Measurements of the optode in the real sample were performed. The potassium content of sheep plasma and whole blood samples determined by the optode were found to agree with the results obtained by ICPMS and ISE methods, providing possibilities for further application in clinical diagnosis.

Table 2. Determination of Potassium Level in Sheep Plasma and Whole Blood Samples Using Upconverting NanorodBased Potassium Selective Optode Compared with Other Methods sample plasma whole blood

ICPMS (mM)

ISE (mM)

found (mM)

recovery (%)

RSD (%)

3.81 26.2

3.8 ± 0.2 29.3 ± 0.6

3.8 ± 0.1 28.3 ± 0.9

99.7 108.0

3.7 3.4

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org. 2621

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Technical Note

(31) Bakker, E.; Simon, W. Anal. Chem. 1992, 64, 1805−1812. (32) Speich, M.; Bousquet, B.; Nicolas, G. Clin. Chem. 1981, 27, 246−248. (33) Kou, L.; Labrie, D.; Chylek, P. Appl. Opt. 1993, 32, 3531−3540. (34) Zhan, Q.; Qian, J.; Liang, H.; Somesfalean, G.; Wang, D.; He, S.; Zhang, Z.; Andersson-Engels, S. ACS Nano 2011, 5, 3744−3757. (35) Zahran, E. M.; Gavalas, V.; Valiente, M.; Bachas, L. G. Anal. Chem. 2010, 82, 3622−3628. (36) Sedlmeier, A.; Achatz, D. E.; Fischer, L. H.; Gorris, H. H.; Wolfbeis, O. S. Nanoscale 2012, 4, 7090−7096.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86(25) 83592562. Fax: +86(25) 83592562. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grants 21135002, 21075062, and 21121091) and the Special-funded Programme on National Key Scientific Instruments and Equipment Development (Grant 2011YQ17006711).

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