Anal. Chem. 2007, 79, 9505-9512
Multiplexed Flow Cytometric Sensing of Blood Electrolytes in Physiological Samples Using Fluorescent Bulk Optode Microspheres Chao Xu,† Katarzyna Wygladacz,† Robert Retter,‡ Michael Bell,‡ and Eric Bakker*,†,§
Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907, Beckman Coulter, Inc., 4300 North Harbor Boulevard, Fullerton, California 92835, and Nanochemistry Research Institute, Department of Applied Chemistry, Curtin University of Technology, Perth, WA 6845, Australia
Polymeric bulk optode microsphere ion sensors in combination with suspension array technologies such as analytical flow cytometry may become a power tool for measuring electrolytes in physiological samples. In this work, the methodology for the direct measurement of common blood electrolytes in physiological samples using bulk optode microsphere sensors was explored. The simultaneous determination of Na+, K+, and Ca2+ in diluted sheep blood plasma was demonstrated for the first time, using a random suspension array containing three types of mixed microsphere bulk optodes of similar size, fabricated from the same chromoionophore without additional labeling. Sodium ionophore X, potassium ionophore III, and grafted AU-1 in poly(butyl acrylate) were the ionophores used in the bulk optode microsphere ion sensors for Na+, K+, and Ca2+, respectively, in combination with the cation-exchanger NaTFPB (sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate) and the same concentration of the chromoionophore ETH 5294 (9-(diethylamino)-5-octadecanoylimino-5H-benzo[a]phenoxazine) in plasticized poly(vinyl chloride). Excellent reproducibility was achieved for the sensing of potassium ions. The effect of sample pH was relatively small at nearphysiological pH and followed theoretical predictions, yet the sample temperature was found to influence the sensor response to a larger extent. Multiplexed ion sensing was achieved by taking advantage of the chemical tunability of the sensor response, adjusting the sensor compositions so that the three types of ion sensors responded with distinct levels of protonation of the chromoionophore. Consequently, three well-resolved peaks were simultaneously observed in the single-channel histogram during the multiplexed calibration as well as in the subsequent measurement of the three cations in 10-fold-diluted sheep plasma. The assigned peak positions corresponded very well to the physiological range of the measured ions. Blood electrolytes such as sodium, potassium, calcium, magnesium, chloride, and phosphate are important for maintaining osmotic gradients, blood pH, hydration of the body, as well as nerve and muscle functions, and their assessment is part of routine clinical diagnostics.1,2 Various techniques can determine blood electrolyte contents, most commonly ion-selective electrodes * Corresponding author. E-mail:
[email protected]. † Purdue University. ‡ Beckman Coulter, Inc. § Curtin University of Technology. 10.1021/ac7016212 CCC: $37.00 Published on Web 11/15/2007
© 2007 American Chemical Society
(ISEs), followed by atomic (flame) emission spectrometry (AES), atomic absorption spectrometry (AAS), as well as fluorimetry and separation methods such as capillary electrophoresis (CE).3-5 ISEs are one of very few methods capable of measuring the free ion activity, i.e., the biologically available fraction from the protein bound or complexed forms. Today, ISEs are widely accepted as the common method for determination of electrolyte ions, and commercial blood analyzers based on ISE principles perform daily in most clinical laboratories. An alternative to ISEs is the bulk optode based on optical transduction,6-8 which uses similar components as an ion-selective membrane, i.e., a polymer matrix containing an ion carrier and a lipophilic ion-exchanger dictating the total extracted ions from the aqueous phase to the membrane phase. An important feature of bulk optodes is the incorporation of a secondary chromogenic ionophore, the so-called chromoionophore, selective to a reference ion (often the hydrogen ion) present at a determined level in the sample. Once the bulk optode is exposed to a buffered sample containing a certain level of the analyte ion, the competitive or collaborative extraction of the analyte ion and the reference ion induces a change in the distribution between the protonated and the unprotonated form of the chromoionophore. The optical signal at equilibrium is indicative of the ion activity of the primary ion. For monovalent cations, the response is indeed the ratio (or the product) of the analyte ion activity and that of hydrogen ions in the sample.9,10 The true two-phase equilibrium for bulk optodes allows the sensor to reach an even lower detection limit compared to ISEs. The response range is conveniently tunable by adjusting the degree of protonation of the chromoionophore at equilibrium, which is a function of the lipophilicity of the ion, the charge, the basicity of the chromoionophore, and the sensor composition as well as the sample pH.10 This unique characteristic of bulk optode (1) Windhager, E. E. Annu. Rev. Physiol. 1969, 31, 117-172. (2) Reinhart, R. A.; Broste, S. K.; Spencer, S.; Marx, J. J., Jr.; Haas, R. G.; Rae, P. Clin. Chem. 1992, 38, 2444-2448. (3) Anker, P.; Jenny, H. B.; Wuthier, U.; Asper, R.; Ammann, D.; Simon, W. Clin. Chem. 1983, 29, 1447-1448. (4) Oesch, U.; Ammann, D.; Pham, H. V.; Wuthier, U.; Zuend, R.; Simon, W. J. Chem. Soc., Faraday Trans. 1986, 82, 1179-1186. (5) Wan, Q. J.; Kuban, P.; Tanyanyiwa, J.; Rainelli, A.; Hauser, P. C. Anal. Chim. Acta 2004, 525, 11-16. (6) Tan, S. S. S.; Hauser, P. C.; Chaniotakis, N. A.; Suter, G.; Simon, W. Chimia 1989, 43, 257-261. (7) Morf, W. E.; Seiler, K.; Rusterholz, B.; Simon, W. Anal. Chem. 1990, 62, 738-742. (8) Seiler, K.; Wang, K.; Bakker, E.; Morf, W. E.; Rusterholz, B.; Spichiger, U. E.; Simon, W. Clin. Chem. 1991, 37, 1350-1355. (9) Bakker, E.; Simon, W. Anal. Chem. 1992, 64, 1805-1812. (10) Bakker, E.; Buhlmann, P.; Pretsch, E. Chem. Rev. 1997, 97, 3083-3132.
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sensors is important and of great advantage in dealing with samples with drastic changes in the analyte ion or with a large pH range.11-13 In recent years, miniaturized forms of the bulk optode sensors have emerged, and microtiter plate based optodes, microbeads and nanospheres have been successfully developed by surface coating, polymerization, sonication, or by a particle caster based on microfluidics.14-18 Smaller amounts of sample and shorter measurement times are required for such sensors. If used as parallel arrays, they can provide highly abundant data that improve the overall accuracy of the measurement and can be used for direct spatial imaging of the analyte.19,20 Monodisperse optical ionsensing microspheres with uniform sizes have been produced with a sonic particle caster in our laboratory for various analytes.15,21 Simultaneous sensing of Na+ and Cl- using microsphere bulk optodes have been achieved by deposition of microspheres that are individually selective to one analyte onto the etched end of an optical fiber bundle.22 Besides blood electrolytes, such sensors are also promising for environmental trace analysis such as lead and silver, with subnanomolar detection limits and short response times.13 Suspension array techniques offer the same advantage of reading a redundant amount of microsensors, but also with better flexibility in the capability of subscreening.23 As a diagnostic tool for the analysis of many biological particles, flow cytometry not only is the standard instrument for the determination of cell size and shape, RNA and DNA contents, and biological membrane characteristics but is also widely used for reading out bead-based assays in immunology, genomics, and proteomics.23 Moreover, polymeric beads of standard sizes are routinely used in flow laboratories for the optical alignment and daily calibration of modern flow cytometry instruments. The availability of various fluorescent labels and numerous receptors, along with the evolution in instrument and software design, has made the suspension array technique a sophisticated yet versatile high-throughput analytical tool with the possibility of sorting and subsampling acquisition. Since the appearance of four-color flow cytometry in the mid 1980s, polychromatic flow cytometry of 11 colors has been reported and has been recently surpassed by 17-color flow cytometry using quantum dots as the fluorescent labels.24,25 Simultaneous interrogation of more than 100 analytes can now be achieved using microbead-based flow cytometry. The resolution (11) Shortreed, M.; Bakker, E.; Kopelman, R. Anal. Chem. 1996, 68, 26562662. (12) Xu, C.; Qin, Y.; Bakker, E. Talanta 2004, 63, 180-184. (13) Wygladacz, K.; Radu, A.; Xu, C.; Qin, Y.; Bakker, E. Anal. Chem. 2005, 77, 4706-4712. (14) Kim, S. B.; Cho, H. C.; Cha, G. S.; Nam, H. Anal. Chem. 1998, 70, 48604863. (15) Tsagkatakis, I.; Peper, S.; Retter, R.; Bell, M.; Bakker, E. Anal. Chem. 2001, 73, 6083-6087. (16) Barker, S. L. R.; Thorsrud, B. A.; Kopelman, R. Anal. Chem. 1998, 70, 100104. (17) Clark, H. A.; Hoyer, M.; Philbert, M. A.; Kopelman, R. Anal. Chem. 1999, 71, 4831-4836. (18) Xu, C.; Wygladacz, K.; Qin, Y.; Retter, R.; Bell, M.; Bakker, E. Anal. Chim. Acta 2005, 537, 135-143. (19) Clark, H. A.; Kopelman, R.; Tjalkens, R.; Philbert, M. A. Anal. Chem. 1999, 71, 4837-4843. (20) Brasuel, M.; Kopelman, R.; Miller, T.; Tjalkens, R.; Philbert, M. A. Anal. Chem. 2001, 73, 2221-2228. (21) Telting-Diaz, M.; Bakker, E. Anal. Chem. 2002, 74, 5251-5256. (22) Wygladacz, K.; Bakker, E. Anal. Chim. Acta 2005, 532, 61-69. (23) Shapiro, H. M. Practical Flow Cytometry, 4th ed.; John Wiley & Sons, Inc.: New York, 2003. (24) De Rosa, S. C.; Herzenberg, A.; Herzenberg, L. A.; Roederer, M. Nat. Med. 2001, 7, 245-248.
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is sufficient for the subscreening of the phenotype of multiple T cells that highly resemble each other in appearance but exhibit distinct biological functions.24,25 In preliminary work, PVC-based ion-sensing fluorescent microspheres, individually selective to potassium or sodium ions, were mixed together and interrogated flow cytometrically for sodium activity in buffer solutions at physiological pH.26 Both types of sensors contained ETH 5294 (excited at 635 nm), and the potassium ion sensing microspheres were labeled with a ringlocked cyanine reference dye that was excited by a second laser at 800 nm. The readout of the response from the two kinds of microspheres maintained the selectivity patterns as previously observed in separate characterizations. These early results motivated us to develop a total optical analytical system for multiplexed sensing of clinical analytes with a flow cytometric platform and various types of microsphere optical sensors. The total number of analytes that can be simultaneously determined on such a system is not only dictated by the uniformity of the microbeads and the design of the instrument but also relies on how well one can anchor the sensing chemistry to the microbeads and maintain spectral resolution among all the fluorescent probes. Here we would like to take initial steps and look into the simplest case of randomly mixing several designed sensors based on the same chromoionophore, each selective to one cation, and to demonstrate the capability of such an optical sensing system for direct ion determination under physiological conditions without additional labeling. This work explores for the first time the parallel assessment of three cations, sodium, potassium and calcium, under physiological conditions by bead-based analytical flow cytometry and includes the measurement of plasma samples. EXPERIMENTAL SECTION Materials. The potassium ionophore III, 2-dodecyl-2-methyl1,3-propanediyl bis[N-[5′nitro-(benzo-15-crown-5)-4′-yl]carbamate] (BME-44), sodium ionophore tert-butylcalix[4]arene tetraacetic acid tetraethyl ester (Na X), the chromoionophore 9-(diethylamino)-5-octadecanoylimino-5H-benzo[a]phenoxazine (ETH 5294), poly(vinyl chloride) (PVC), and bis(2-ethylhexyl) sebacate (DOS) were purchased from Fluka (Milwaukee, WI). The calcium ionophore AU-1 covalently attached to poly(n-butyl acrylate) was prepared as described.18 The ion-exchanger, sodium tetrakis-[3,5bis(trifluoromethyl)phenyl]borate (NaTFPB), was purchased from Dojindo Laboratories (Gaithersburg, MD). Dichloromethane (DCM), ethyl acetate, and xylenes were obtained from Fisher Scientific. Cyclohexanone (99.8%) (Aldrich), tetrahydrofuran (THF) (Fluka), poly(ethylene glycol) (PEG) (Polysciences, Inc.) were ACS grade. Tris(hydroxymethyl) aminomethane (Tris) was reagent grade from Sigma Aldrich. Salts, acids, and bases of the highest available quality were used. All standard solutions were prepared with freshly deionized water (18.2 MΩ cm-1) using a Nanopure (Millipore) water purification system. The sheep plasma sample was obtained from Hemostat, CA and was stabilized with heparin. Preparation of Bulk Optode Fluorescent Microspheres. Polymeric microsphere bulk optodes were prepared according to earlier reported procedures.15,22 A total mass of 45 mg of the sensor components was dissolved in 1.25 mL of cyclohexanone and subsequently diluted with 50 mL of DCM or ethyl acetate. A 500 (25) Chattopadhyay, P. K.; Price, D. A.; Harper, T. F.; Betts, M. R.; Yu, J.; Gostick, E.; Perfetto, S. P.; Goepfert, P.; Koup, R. A.; De Rosa, S. C.; Bruchez, M. P.; Roederer, M. Nat. Med. 2006, 12, 972-977. (26) Retter, R.; Peper, S.; Bell, M.; Tsagkatakis, I.; Bakker, E. Anal. Chem. 2002, 74, 5420-5425.
Figure 1. Schematic setup of the optical multiplexed ion-sensing system based on an analytical flow cytometer and fluorescent ion-sensing bulk optode microsphere sensors fabricated with PVC-DOS. A 635 nm red laser was used as the excitation source for the fluorescent microsphere bulk optode sensors.
µL aliquot of xylenes was then added to the diluted cocktail. The resulting polymer cocktail was filtered through a 0.45 µm nylon syringe filter to remove any solid impurities. All original cocktails contained PVC and DOS (1:2 by mass) as the matrix. The rest of the compositions were as follows: K+-selective microsphere bulk optodes, 11.8 mmol/kg BME-44, 3.0 mmol/kg ETH 5294, 5.8 mmol/kg NaTFPB; Na+-selective microsphere bulk optodes, 11.9 mmol/kg Na X, 2.9 mmol/kg ETH 5294, 5.8 mmol/kg NaTFPB; Ca2+-selective microsphere bulk optodes, 15.2 mmol/kg calculated concentration of AU-1 grafted at 5 wt % in poly(n-butyl acrylate), 2.8 mmol/kg ETH 5294, 5.0 mmol/kg NaTFPB. Ion-sensitive microspheres were prepared using a particle caster, which consists of a mixing chamber assembly with a ceramic orifice.15 Two streams (polymer core stream and deionized water sheath) were directed to the mixing chamber, and the polymer stream was ejected from the orifice. A piece of piezoelectric transducer oscillated at adjusted frequency and resulted in the formation of polymer droplets, which were collected directly in 25 mL glass vials. A surfactant (3% (v/v) PEG) was added continuously to the droplet stream to minimize clumping. After casting, the microspheres were stored in the dark. Once the organic solvent had evaporated from the polymer droplets, polymeric particles of about 10-12 µm were obtained. For microscopy measurements, after the microspheres were cured for at least 24 h, a small amount of the particle slurry was transferred to a glass slide, and the whole slide was immersed in deionized water in the dark until incubation with the sample solution. Sample Preparation and Data Acquisition. All samples were prepared with 10 mM Tris, and the pH was adjusted with 1 M HCl. The fully protonated and deprotonated states of the chromoionophore were characterized with 10 mM HCl and 10 mM NaOH, respectively. Artificial blood solutions (ABS) mimicking the blood electrolyte level in 10-fold-diluted blood originally contained 10 mM NaCl, 0.1 mM of MgCl2, 0.2 mM CaCl2, and 0.5 mM KCl in 10 mM Tris buffer at physiological pH.27 This recipe was followed for the preparation of standard solutions for the single calibrations as well as the multiplexed calibrations, except that the composition of the analyte ion was replaced with various levels according to the calculated measuring range of each sensor. The sheep plasma sample was centrifuged for 5 min or filtered to remove excessive particles, followed by the dilution to 10-fold or 50-fold with a 10 mM Tris buffer, and the pH was adjusted to 7.4, or other designated values. (27) Speich, M.; Bousquet, B.; Nicolas, G. Clin. Chem. 1981, 27, 246-248.
Selectivity coefficients were evaluated by the separate solutions method (SSM) by comparing the fluorescence response for the primary and interfering ions at pH 7.4 at R ) 0.5.9 For fluorescence microscopy, approximately 20 mL of the sample buffer was used for the equilibration with the microsphere sensors for 10-15 min. For flow cytometry measurements, 0.9 mL of sample buffer was added to the sample vial, followed by the addition of 0.1 mL of the microsphere slurry. The ion activities were recalculated to reflect the actual composition in the final solution. The microspheres were incubated in the sample solution for 30 s before the readout with flow cytometry. For evaluating the pH effect, 10 mM Tris buffers containing various potassium levels were prepared in triplicates, and the pH was adjusted to 7.35, 7.4, and 7.45 with HCl. For the measurement at different temperatures, the flow cytometry sample tubing was surrounded by a water jacket connected to a reservoir where water at a designated temperature was constantly recirculated. The sample tubes filled with solutions were equilibrated with the water at the designated temperature for at least 10 min prior to each measurement. Records on the temperature were taken before and after each run, and the average value was used in the final presentation. For multiplexed sensing, a total of 0.1 mL of slurry containing all three microspheres was added to the 0.9 mL of standard solution or the 10-diluted plasma sample at physiological pH. All the standard buffers contained Na+, K+, Mg2+, and Ca2+. Each contained a constant background of the three interfering ions at physiological levels mimicking the 10-fold-diluted plasma, while the primary ions were 10-4 to 10-2 M K+, 10-3 to 10-1 M Na+, or 10-4 to 10-2 M Ca2+. The microspheres were subsequently read out by flow cytometry under the same condition after a 60 s incubation period. Optical Measurements. For the fluorescence microscopy measurements, a PARISS imaging spectrometer (Light Form, Belle Mead, NJ) was used in combination with a Nikon Eclipse E400 microscope to characterize the microspheres as described previously.22 The exposure time for the fluorescence data acquisition was kept at 200 ms. Ratiometric measurements were performed by comparing the fluorescence emission of ETH 5294 at 648 and 679 nm, respectively. Each microsphere was used once for the recording of the spectra. Flow cytometry experiments were carried out with a modified EPICS XL flow cytometer (Beckman Coulter)26 as illustrated in Figure 1. The instrument uses a laser excitation source at 635 nm. Fluorescence emitted between 650 and 675 nm is collected with a 650 nm long-pass emission filter and a 660 ((15) nm band-pass filter. Analytical Chemistry, Vol. 79, No. 24, December 15, 2007
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RESULTS AND DISCUSSION The bulk optode microsphere sensors were doped with a cation-exchanger R-, a neutral ionophore L (forming 1:n complexes with the analyte ion), and a neutral H+-selective chromoionophore Ind.10 For such bulk optodes, the cationic analytes Iz+ with a charge of z+ are extracted from the aqueous solution into the organic sensing phase, thereby expelling protons from the organic phase via a cation-exchange mechanism:8
Iz+(aq) + nL(org) + zIndH+(org) + zR-(org) ) ILnz+ (org) + zInd(org) + zR-(org) + zH+(aq) (1) Combining charge and mass balances and defining the mole fraction of protonated Ind as (1 - R), the activity of the analyte may be expressed as:10
aI ) (zKex)
RT- - (1 - R)IndT z R a 1 - R H {L - (R - - (1 - R)Ind )(n/z)}n T T T
(
-1
)
(2) The total concentrations of chromoionophore, ionophore, and ion-exchanger are labeled as IndT, LT, and RT-, respectively, and Kex is the ion-exchange constant describing eq 1. As indicated by eq 2, cation-selective bulk optodes based on neutral carriers that are described in this work indeed respond to the ratio of the activities of analyte ion and the proton in the sample solution. In a buffered solution where the pH is known, the activity of analyte ion can be determined by the degree of protonation of the chromoionophore (1 - R), which is calculated based on the observed emission intensities for the protonated and unprotonated form of the chromoionophore. For a specific analyte ion, the dynamic range of the bulk optodes can usually be tuned by adjusting the pH of the sample, the chromoionophore structure for different basicity, the sensing matrix, or the concentration of sensing components. This unique flexibility is used here to design bulk optode sensors for various sample analyte concentrations and is quite beneficial for the development of multiplexed assays. A modified EPICS XL flow cytometer (illustrated in Figure 1) was used here for the interrogation of the microsphere sensor response. Particles were separated and aligned by the sheath liquid, guided through the flow cell, and optically excited by the laser. Scattering and fluorescence signals were collected for each particle, and the counts were based on the observed fluorescence intensity, forming a fluorescence channel histogram where the accumulated counts were plotted versus the peak channel fluorescence. The widely studied chromoionophore ETH 5294 was used here for the three types of bulk optodes. When excited at 510 nm (fluorescence spectroscopy), this fluorescent pH indicator shows dual emission at 647 and 679 nm, corresponding to the unprotonated and protonated form, respectively.13 When excited with the laser (635 nm) on the EPICS-XL flow cytometer, the deprotonated form of ETH 5294 becomes spectrally muted; therefore, the chromoionophore exhibits a single emission peak corresponding to the protonated form at 679 nm.26 This translates into an inverse correlation between the cation activity and the fluorescence intensity. As the sample ion concentration increases, the fluores9508
Analytical Chemistry, Vol. 79, No. 24, December 15, 2007
Figure 2. Fluorescence microscope image of potassium-selective microspheres using ETH 5294 as the chromoionophore. The microspheres were mass produced by a particle caster, with an estimated average size of ∼11 µm.
cence intensity from the protonated form of ETH 5294 decreases because more protons are expelled from the polymer phase. The resulting shift in the peak position in the single-parameter histogram reflects the ion response. Assuming bulk optode behavior of the fabricated microspheres, the mole fraction of unprotonated form, R, can be expressed by the fluorescence peak position (P) in the single-parameter histogram from the FL1 channel as follows:
R)
Ppro - P Ppro - Pdep
(3)
where Ppro and Pdep are the peak position obtained at the fully protonated or unprotonated condition of the chromoionophore, respectively.18 Microsphere bulk optodes that are individually selective to potassium, sodium, and calcium were generated by the particle caster and subsequently characterized by fluorescence spectroscopy. Figure 2 shows the fluorescence images of the generated potassium-selective microspheres with an average diameter of about 11 µm. The particles appeared to be spherical, with a smooth surface and uniform size. Calibration curves obtained with fluorescence spectroscopy in separate 10 mM Tris buffer solutions exhibited a potassium response between 10-4 to 10-2 M, with very good selectivity toward common interferences in blood, such as sodium, calcium, and magnesium. For the theoretical curve (eq 2) of the potassium response, log Kex ) -4.35 was used to fit the data with the theoretical prediction. Calibration curves were obtained from flow cytometry measurements, with shorter incubation times (∼1 min) in order to minimize the clumping of the particles in the sample. A histogram with particle counts versus fluorescence peak was obtained for each sample concentration, and the resulting overlay is shown in Figure 3 (solid lines). The peaks appeared to be narrow and showed a clearly distinct fluorescence peak position on a scale from about 10 to near 100, for different potassium ion activity in the sample buffer. The histograms also indicated that the sensing range of the potassium ion lies in the range of 10-4 to 10-2 M at that pH. The entire histogram from about 3000 to 10 000 of such microspheres normally has coefficients of variation (CV) from 10.69 to 3.87 that
Figure 3. Overlay of the cytometry histograms for the response of the potassium-selective bulk optode microspheres toward various potassium ion activities (pH ) 7.40). Histograms represented by solid lines were obtained from 10 mM Tris buffers (K+ from 10-5 to 10-1 M), while the one labeled with a dashed line was obtained from 10fold-diluted sheep blood plasma. Table 1. Required Logarithmic Selectivity Factors log Osel KIJ (Required) for Ion-Selective Microsphere Optodes Assuming Interfering Ion Levels in 10-Fold-Diluted Blood Plasma (pH ) 7.40)a
Figure 4. Calibration curve of the potassium-selective microspheres by flow cytometry with a constant background of interfering ions in the artificial blood solution (ABS), followed by the measurement of 10-fold-diluted blood plasma and standard addition to 50-fold-diluted plasma. All data were obtained at pH ) 7.40. The data points for the calibration (filled circles) were obtained from the histograms as typically shown in Figure 3. Selectivity data were obtained with the separate solutions method (SSM). The open circles denote the data points for the standard addition with spikes of known potassium concentrations. The squares are the observed response of the sensor toward a single sample of 10-fold-diluted plasma.
can be further lowered when a larger population of the microspheres is sampled.26 PVC-based bulk optode microsphere sensors become quite attractive in measuring ion activities when hyphenated with suspension array technology. The readout speed is greatly improved (20-100 particles/s, and theoretically orders of magnitude larger than this) compared to the fluorescence spectroscopy of single microspheres. The shorter data acquisition time helps avoid the variance in the signal due to sample drying and ensures negligible risk of dye photobleaching. The above data were used to calculate the degree of protonation at each sample concentration according to eq 3. A calibration curve derived from eq 2 was used to fit the experimental data (with log Kex ) -4.4 for K+). SSM was used for the selectivity measurement with flow cytometry as well. The experimental selectivity coefficients (log kOsel values9) for potential interferIJ ences from both methodologies are listed in Table 1, and both are compared to the calculated required selectivity coefficients based on their average levels in human blood.4,28 It was found that the two characterization methods agree with each other, not only in the response characteristics but also in the selectivity pattern. For both calibrations, at half-protonation of the chro-
moionophore (R ) 0.5), the corresponding potassium activity was about 0.5 mM, which is close to the normal range of potassium ion activity in 10-fold-diluted blood sample.3,29 Because of the abundant concentrations of common interferences for potassium, it is advisable to calibrate the sensor in a fixed background of interferences according to the composition of ABS, which mimics 10-fold-diluted blood samples (pH 7.4). The K+-selective fluorescent microsphere bulk optodes were calibrated in such sample buffers, and a new calibration curve is shown here in Figure 4. Each data point is marked as a filled circle, and the theoretical curve for the sensor response is shown with log Kex ) -4.30 for K+. The selectivity data obtained from the ABS (FIM and SSM) were listed in Table 2, together with the fluorescence spectroscopy data. The satisfactory response toward K+ and the close value of Kex demonstrated that the bulk optode microsphere sensors that are tested here were capable of maintaining sufficient selectivity in a background of blood electrolyte composition and were suitable for physiological measurements. Subsequently, flow cytometry measurements of potassium activity were performed in diluted and buffered sheep plasma samples (50- or 10-fold dilution) at physiological pH with the calibrated K+-selective fluorescent microsphere optodes, followed by standard addition with potassium solution of known concentrations at the same pH. The measurement for 10-fold-diluted sheep plasma was performed in triplicates, and the data points were obtained from the standard addition are summarized in Figure 4, denoted by square symbols. The calculated average of the potassium activity in the sheep plasma sample before dilution is 6.56 ( 0.27 mM. The potassium activities in the original sheep blood plasma is determined as 7.29 ( 0.29 mM, considering that the plasma sample was spiked with a heparin solution as an anticoagulant at an ∼1:9 ratio by volume. Aliquots of buffer solutions with known potassium concentrations were spiked separately into the 50-fold-diluted plasma sample
(28) Eugster, R.; Rusterholz, B.; Schmid, A.; Spichiger, U. E.; Simon, W. Clin. Chem. 1993, 39, 855-859.
(29) Wang, K.; Seiler, K.; Morf, W. E.; Spichiger, U. E.; Simon, W.; Lindner, E.; Pungor, E. Anal. Sci. 1990, 6, 715-720.
interference primary ion K Na Ca
KOsel IJ
K
log Na
-3.39 -0.27 -2.31
-3.79
(required) Ca -1.30 +0.35
Mg -1.22 +0.43 -1.62
a The required log KOsel values were calculated according to ref 9 IJ assuming the separate solutions method (SSM) and a 2% error in the sensor response. R ) 0.5 was used in the calculation of all coefficients. The ion activity data for the involved blood electrolytes were obtained from refs 4 and 27.
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Table 2. Observed Exchange Constants and Selectivity Factors for Potassium-Selective Microsphere Optodes in Fluorescence Spectroscopy and Flow Cytometry (in Standard Buffer Solutions, pH 7.4)a fluorescence spectroscopy SSMb ion
log Kexch
K Na Mg Ca
-4.40 -8.50 -16.4 -17.6
flow cytometry FIM-ABSc
SSM log
kOsel IJ
0 -4.10 -4.17 -5.37
log Kexch log -4.40 -8.00 -16.00 -19.50
kOsel IJ
0 -3.65 -3.82 -7.32
log Kexch log kOsel IJ -4.30 -8.60 -16.10 -17.0
0 -4.30 -3.98 -4.88
a The stoichiometries of the complex formation between the ionophore and the primary ion were assumed as 1:1, 1:1, and 1:3, respectively, for the sodium, potassium, and calcium bulk optodes described here. b For the selectivity data with SSMs, each solution contained 10 mM Tris and a total concentration of 0.5 or 1 M of the interfering ion. c For the sensor responses in the fixed background of interfering ions, an artificial blood solution (ABS) was prepared with a mixture of all involved interfering ions mimicking the composition of 10-fold-diluted blood plasma. This solution was used as the background for the preparation of the standard potassium solutions.
Figure 5. Sensor response of the potassium-selective microspheres toward the ratio of the activities of potassium over proton. The data points were obtained from buffer solutions with identical potassium contents but different pH of 7.35, 7.40, and 7.45, each denoted as squares, filled circles, and triangles, respectively.
buffers (pH 7.4). The calculated potassium level for the 50-folddiluted plasma sample was first calculated based on the location of the fluorescence peak in the FL1 histogram, and the obtained value was used for the actual potassium concentration and ion activity in the solutions after each standard addition (shown in Figure 4, open circles). The data points for the standard addition were on the calibration curve, showing that the flow cytometric readout for the potassium-selective microspheres were capable of recognizing a concentration change as small as 2 × 10-5 M that was added to the 50-fold-diluted physiological sample. The response of the bulk optode sensor depends on the sample pH.6 For a monovalent ion, bulk optode theory (eq 2) predicts that a change of 0.1 pH units should induce a similar deviation for the logarithmic ion activity30 because of the direct correlation of the sensor response between the sample pH and the ion activity of the analyte. Since the normal variance in the level of the blood electrolytes are quite narrow, such influence in the sample pH may play a significant role in correctly assigning the signal to the actual value for the daily calibrations. We prepared here buffer samples containing precisely the same potassium activity but with slightly different pH values (7.35 and 7.45). The calibration curves for flow cytometry were generated with the incubation to such modified samples compared to that obtained from buffers at 7.40, as presented in Figure 5. The degree of protonation here is plotted against the logarithmic value of the ratio between the activity of potassium ion and proton. According to bulk optode theory, this ratio is a function of the sensor composition and the property of the ionophore and the ion but is sample independent. From Figure 5, it is clear that the almost overlapping data points at different ratios confirm theoretical predictions. The response from potentiometric sensors using ISEs is temperature-dependent and result in changes in the slope that require recalibration and careful temperature control.4 A temperature influence on the selectivity of ion-selective membranes was also reported for certain matrices.31 Ionophore-based sensors are
mostly fabricated with dissolved sensing components in polymers that are essentially highly viscous liquids to ensure flexibility of the sensing components for the reversible binding with the analyte ion. One of the important requirements for an ideal polymer matrix to be used for ion-selective membranes or bulk optodes is that the glass transition temperature (Tg) should be lower than the temperature at which the sample is measured, typically room temperature.10 Microsphere bulk optode ion sensors fabricated from plasticized PVC were here tested with regard to temperature sensitivity, to our knowledge for the first time. The fluorescent microsphere ion sensor for potassium ions was taken as an example. Samples were pre-equilibrated in a water bath at the designated temperature, and the sample tube was maintained at approximately the same temperature using a circulating water jacket during the flow cytometry readout. The results are plotted in Figure 6, with the peak position ranging from lower than 20 to as high as 40 with the change of temperature from 5 to 35 °C. Although the results shown here are preliminary and are limited by the insulation of the sample tube from the surroundings, the temperature of the sample appears to influence
(30) Lerchi, M.; Bakker, E.; Rusterholz, B.; Simon, W. Anal. Chem. 1992, 64, 1534-1540.
(31) Oosaki, S.; Kawai, Y.; Yajima, S.; Kimura, K. Anal. Sci. 2004, 20, 11651169.
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Figure 6. Observed peak position from the histograms obtained at different temperature of the 10-fold-diluted plasma sample. The sample tube was constantly equilibrated with a flowing water bath (5-40 °C) in a surrounding jacket during the data acquisition. The plotted sample temperature was the average value before and after the cytometry measurement.
the response of the tested microsphere bulk optode sensors characterized by flow cytometry, especially above 25 °C. Since the extent of the temperature dependence is difficult to theoretically predict, careful temperature control is recommended for routine applications. The goal of this study was to develop a flow cytometry assisted multiplexed assay for the direct readout of blood electrolyte activities using mixed bulk optode microsphere sensors. Microsphere bulk optodes that are selective to potassium, sodium, and calcium were fabricated, randomly mixed, and incubated with buffered physiological samples to interrogate the activity of each analyte in a single reading. Multiple fluorescent labels are certainly advantageous to ensure accuracy of multiplexed assays but require more complex instrumentation. We therefore set out to explore whether it is feasible to use a single fluorescent probe for the multiplexed assay of three different ions. If the fluorescent beads may be fabricated with a narrow size distribution and uniform doping efficiency, as with the particle caster used here, overlapping peak positions may be more easily resolved. In principle, each type of sensing microsphere could be prepared at a different size to give distinguishable fluorescence intensities. Here, however, we took advantage of the tunability of the dynamic measuring range of bulk optode sensors. Each type of microsphere sensor was fabricated to respond to the analyte in the physiological range at distinct regions of the level of protonation of the chromoionophore. This should make them recognizable in the histogram obtained from the fluorescence detector. The three common cations of interests, Na+, K+, and Ca2+, have normal concentration ranges of 135-150, 3.5-5.0, and 1.01.2 mM in undiluted human plasma, respectively.1,4 The composition of each ion sensor was carefully adjusted according to Kex values for the primary ion in PVC-DOS matrix previously determined in the literature.22,26,32 The sensing compositions were optimized according to bulk optode theory and eq 2, to reach a final composition in each sensor with a well-resolved variance in the expected value of (1 - R), between each adjacent data point (composition listed in the Experimental Section) assuming the doping of the same concentration of chromoionophore in each sensor. The microspheres containing the same concentration of ETH 5294 were prepared using the optimized composition and were separately calibrated using flow cytometry (see Figure 7A). The log Kex values for each type of sensor and their primary ions were found as follows: -4.3 for potassium (filled circles), -17.0 for calcium (half-filled circles), and -5.0 for sodium (open circles), respectively. The (1 - R) values were calculated as 0.55 for potassium, 0.35 for calcium, and 0.15 for sodium, respectively, for the expected ion activities in a 10-fold-diluted plasma sample at physiological pH. This would suggest sufficient resolution for the approach used here. Calibrations were performed with a random mixture of particle slurry containing all three types of microspheres. For each analyte, three standard sample solutions were used, each containing various primary ion levels but with a constant background of the other two ions involved in this study at corresponding physiological levels. Three fluorescent peaks were simultaneously formed (32) Qin, Y.; Peper, S.; Radu, A.; Ceresa, A.; Bakker, E. Anal. Chem. 2003, 75, 3038-3045.
Figure 7. (A) An overlay of the response curves for the three optical sensors described in this study, each curve obtained from a calibration using single sensors. (B) An overlay of the sensor response curves obtained from a multiplexed calibration using a random mixture containing all three sensors. Inset: A histogram showing three accumulated peaks generated by the randomly mixed optode microspheres upon the equilibration with a 10-diluted-sheep plasma sample at physiological pH.
on the histogram, indicating a resolved response from all three types of fluorescent microsphere ion sensors. Ideally, changing the activity of only one ion would cause a shift in just one of the three fluorescent peaks, while the other two should stay in the same position. For two types of microspheres whose accumulated fluorescence peaks are adjacent to each other, however, peak separation became less distinct as a result of the partially overlapping degree of protonation of the individual sensing particles. Despite making quantification more difficult, identification of the shifting peak was still possible, and the change in peak position was consistent with the change in the ion activity in the sample. The positions of the peaks were translated to degrees of protonation and plotted in a new mixed particle calibration curve, see Figure 7B. The log Kex values for each type of sensor and their primary ions were now found as -4.35 for potassium (filled circles), -17.3 for calcium (half-filled circles), and -4.9 for sodium (open circles), respectively. Little difference was noted between the single calibration and the multiplexed calibration (Figure 7, parts A and B). The resemblance also indicated that the moving peaks in the multiplexed calibration were assigned correctly to the change of the ion activity. The errors generated by the interfering ions (the two measured cations and magnesium ion) in the mixed background were nearly negligible (∼1%). This demonstrated that the selectivity of each microsphere was preserved in this multiplexed assay. Analytical Chemistry, Vol. 79, No. 24, December 15, 2007
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With the use of the three types of the microspheres characterized above, a label-free multiplexed ion sensing using flow cytometry was achieved and shown in the inset of Figure 7B, with the histogram obtained for the mixed bulk optode microspheres. Three peaks are clearly visible, with the peak position at 9.03, 17.3, and 41.7, corresponding to the signals from the microsphere sensors for sodium, calcium, and potassium, respectively. The peak position in this histogram was used to calculate the degree of protonation and, subsequently, the ion activity that was originally in the 10-fold-diluted sample. The ion activity of each analyte from the flow cytometry is plotted in Figure 7B as the open squares on the calibration curve. The dotted lines indicate the degree of protonation observed for each analyte and the corresponding ion activity. The ion activities in the 10-fold-diluted plasma sample at pH 7.4 were determined as 13.1 mM (sodium), 0.24 mM (calcium), and 0.33 mM (potassium) in the diluted sample (0.9 mL of 10-fold-diluted plasma mixed with 0.1 mL of the mixed particle slurry). The activities of the three ions in the original undiluted sheep plasma sample were determined as 126 mM (sodium), 1.52 mM (calcium), and 3.62 mM (potassium), which correspond well to the physiological values. The (1 - R) values for the three different types of sensors were found as 0.62, 0.39, and 0.15 for potassium, calcium, and sodium, respectively, with a nearly equal distance of around 0.24 between two neighboring data points. This indicates that the involved sensing chemistry is able to be finely tuned according to the nature of the sample and can be adapted quite readily to bulk optode microspheres for other ions. The three fluorescence peaks in Figure 7B exhibit partial overlap, which implies that there is a need to further improve the resolution of the peaks. Sources of errors for this method include the uniformity of the size distribution of the microsphere sensors, the loading efficiency of the sensing components in each sensor, as well as instrument factors. The use of fluorescence labels, multiple light sources, and customized
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software may also help optimize this technique for practical clinical analysis. CONCLUSIONS In this work, we studied the methodology for the direct measurement of common blood electrolytes in physiological samples using bulk optode microsphere sensors read by flow cytometry and demonstrated for the first time the feasibility of simultaneous determination of multiple analytes using a random suspension array of three types of mixed microsphere ion sensors fabricated from the same chromoionophore without the need of additional labeling. The fluorescence signals accumulated in three distinct regions in the fluorescence histogram that corresponded to the desired range of each analyte ion. This was achieved by carefully tuning the sensor composition to shift the responses of each involved sensor to distinct regions in the fluorescence histogram so that the sensors were sorted by their responses toward each analyte. Fast, stable, and clear fluorescence peaks were obtained simultaneously, and the assigned peak position corresponded very well to the physiological range of the ions that were measured. The simultaneous reading of several analytes has multifold advantages, allowing us to simplify existing laboratory procedures and shortening the assay time with easily replaceable sensing chemistry. The capability of the optical sensing platform demonstrated here can certainly be further expanded by additional fluorescence labeling and narrower size distribution of the microspheres, as well as the incorporation of multiple fluorescence channels and laser sources for more complex sensing tasks for a bead-based suspension array total clinical analysis system. ACKNOWLEDGMENT The authors thank Beckman Coulter, Inc. for supporting this research. Received for review July 31, 2007. Accepted October 7, 2007. AC7016212
