Anal. Chem. 2003, 75, 525-529
Microfabricated Electrophoresis Chip for Bioassay of Renal Markers Joseph Wang* and Madhu Prakash Chatrathi
Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003
A novel capillary electrophoresis chip-based detection system for simultaneous measurements of the renal markers creatine, creatinine, p-aminohippuric acid, and uric acid is described. Fluid control is used for mixing the sample with the enzymes creatininase (CA), creatinase (CI), and sarcosine oxidase (SOx) and for separating the neutral hydrogen peroxide end product from the anionic p-aminohippuric and urate species. The ‘total’ (creatinine and creatine) signal was measured with the running buffer containing all three enzymes, while the creatine signal alone was recorded by mixing the sample with the CISOx solution. Creatinine concentrations are measured by comparing the response in the presence and absence of CA. The peroxide product and the oxidizable p-aminohippuric and uric acids are detected electrochemically at a downstream gold-coated thick-film amperometric detector. The four renal markers are readily measured within 5 min, while creatinine/creatine within less than 2 min. Factors influencing the performance, including the level of three enzymes, separation voltage, and detection potential, are optimized. Applicability to urine samples is demonstrated. Such a multianalyte microchip detection device would allow renal function testing to be performed more rapidly, easily, and economically in the point-of-care setting. Microfluidic devices (also referred to as “labs-on-chips”) have shown exciting possibilities in biochemical analysis.1,2 The ability to perform all the steps of a biological assay on a single platform offers significant advantages in terms of speed, performance, reagent consumption, miniaturization, cost, and automation. Such advantages have been documented in connection with chip-based enzymatic assays,3,4 immunoassays,5,6 and nucleic acid detection.7,8 The ability to conduct enzymatic assays on microchip platforms * Corresponding author. E-mail:
[email protected]. (1) Figeys, D., Pinto, D. Anal. Chem. 2000, 71, 330A-335A. (2) Kutter, J. P. Trends Anal. Chem. 2000, 19, 352-363. (3) Wang, J. Electrophoresis 2002, 23, 713-718. (4) Hadd, A. G.; Raymond, D. E.; Halliwell, J. W.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, 3407-3412. (5) Sato, K.; Tokeshi, M.; Kimura, H.; Kitamori, T. Anal. Chem. 2001, 73, 12131218. (6) Wang, J.; Ibanez, A.; Chatrathi, M. P.; Escarpa, A. Anal. Chem. 2001, 73, 5323-5327. (7) Righetti, P. G.; Gelfi, C.; D, Acunto, M. R. Electrophoresis 2002, 23, 13611374. (8) Fan, Z. H.; Mangru, S.; Granzow, R.; Heaney, P.; Ho, W.; Dong, Q.; Kumar, R. Anal. Chem. 1999, 71, 4851-4859. 10.1021/ac020560b CCC: $25.00 Published on Web 12/20/2002
© 2003 American Chemical Society
has been exploited for rapid measurements of glucose9 or amino acids10 or acetylcholinesterase inhibitors4 in connection with oncolumn, precolumn, or postcolumn biocatalytic reactions, along with microchip separations of the products or substrates. There is an increasing demand for simple, reliable, and inexpensive assays of metabolites that are indicators of health status.11 An example is assessment of creatinine, creatine, uric acid, urea, and p-aminohippuric acid in biological fluids, which are factors in the evaluation of renal and muscular functions.12 Renal function tests fall into three categories: (1) tests predominantly of glomerular function, (2) tests reflecting severe glomerular or tubular damage (or both), and (3) tests of predominantly tubular function.13 Therefore, determination of glomerular filter rate (GFR) and effective renal plasma flow (ERPF) is essential. Creatinine is the end product of creatine metabolism,14 and its clearance level is a good indicator in the diagnosis of renal disease, thyroid malfunction, and muscular disorders. While serum and urine levels of urea and creatinine are used as good markers, p-aminohippuric acid clearance is used for the measurement of ERPF.15 Unlike urea, the concentration of creatinine is not influenced by the protein intake and therefore it is a more reliable indicator of renal function.16 Creatine measurement in blood is a valuable index of muscular disorder. Urine levels of creatinine and uric acid are good indicators of GFR of the kidney, the amount of fluid filtered per unit time. Uric acid also serves as a marker for tubular reabsorption of nephrons, and its elevated levels indicate gout, renal disease, and higher rate of nucleic acid breakdown. The level of p-aminohippuric acid in urine is an indictor of the tubular secretion of the nephrons and of the amount of plasma flow to the kidneys per unit time.15 The search for analytical tools for measuring renal markers has prompted the development of various enzyme-based biosensors,17,18 chromatographic19,20 or electrophoretic21,22 protocols, or (9) Wang, J.; Chatrathi, M. P.; Tian, B. M.; Polsky, R. Anal. Chem. 2000, 72, 2514-2518. (10) Wang, J.; Chatrathi, M. P.; Iba´n ˜ez, A.; Escarpa, A. Electroanalysis 2002, 14, 400-404. (11) Tudos, A. J.; Besselink, G. J.; Schasfoort, R. B. M. Lab Chip 2001, 1, 8395. (12) Burtis, C. A., Ashwood, E. R., Eds. Tietz Textbook of Clinical Chemistry, 2nd ed.; Saunders: Philadelphia, 1994. (13) Ravel, R. Clinical Laboratory Medicine: Clinical Application of Laboratory Data, 5th ed.; Mosby Year Book: St. Louis,, MO, 1989; Chapter 13. (14) Narayanan, S.; Appleton, H. D. Clin. Chem. 1980, 26, 1119-1126. (15) Henry, J. B. Clinical Diagnosis and Management, 16th ed.; Saunders: Philadelphia, 1979; Chapter 6. (16) Kazmlerczak, S. C. Anal. Chem. 1991, 63, 173R-176R. (17) Tsuchida, T.; Yoda, K. Clin. Chem. 1983, 29, 51-55. (18) Madaras, M. B.; Buck, R. P. Anal. Chem. 1996, 68, 3832-3839.
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Figure 1. Layout of the microchip for bioassays of renal markers. (A) Assay of creatine (Cre), uric acid (UA), and p-aminohippuric acid (pAH) (B) Assay of creatinine (Crn)/Cre, UA, and pAH. The individual injection and separation steps are shown in each case. Bioassay A is carried out with a running buffer (R2) containing the enzymes creatinase (CI) and sarcosine oxidase (SOx), while assay B is performed by mixing the sample with the creatininase (CA)-CI-SOx solution (R1). The sample (S) solution is a mixture of creatine, creatinine, p-aminohippuric acid, and uric acid. See text for more details.
the common Jaffe-based spectrophotometric method.23 Amperometric biosensors17,18 have commonly relied on the coimmobilization of the three enzymes [creatininase (CA), creatinase (CI), sarcosine (SOx)] in connection with the biocatalytic reaction sequence CA
creatinine + H2O 98 creatine Cl
creatine + H2O 98 sarcosine + urea SOx
sarcosine + H2O + O2 98glycine + HCHO + H2O2
(1) (2) (3)
There are no reports of chip-based microsystems for monitoring renal function markers other than uric acid.9,24 In this report, we describe a new chip-based protocol, based on the coupling of enzymatic bioassays and electrophoretic separations, for rapid and simultaneous measurements of major renal markers. The new micromachined analyzer couples on-chip enzymatic reactions of creatinine and creatine with a CE separation of p-aminohippuric and uric acids (Figure 1). For this purpose, the sample mixture and the enzymes (CA, CI, SOx) are mixed, the enzymatically liberated neutral peroxide species is separated from the anionic urate and p-aminohippuric acids in the separation/reaction channel, and the three species are detected at the end-column amperometric detector. The “total” (creatinine + creatine) signal is obtained with a running buffer containing all three enzymes (Figure 1B), while the creatine response is recorded without the creatininase enzyme (Figure 1A). Such biochip operation results in an attractive performance that compares favorably with that of conventional biosensors or traditional analyzers. The operating conditions and benefits of the new “renal markers” biochip are discussed below. EXPERIMENTAL SECTION Reagents. Sarcosine oxidase (29 units mg-1, from Bacillus species), creatinase (12.8 units mg-1, from Actinobacillus species), (19) Kock, R.; Sitz, B.; Delvoux, B.; Greiling, H. Eur. J. Clin. Chem. Clin. Biochem. 1995, 33, 23-29. (20) Brown, N. D.; Sing, H. C.; Neeley, W. E.; Koetitz, S. E. Clin. Chem. 1977, 23, 1281-1283. (21) Burke, D. G.; MacLean, P. G.; Walker, R. A.; Dewar, P. J.; Smith-Palmer, T. J. Chromatogr., B 1999, 732, 479-485. (22) Clark, E. A.; Fanguy, J. C.; Henry, C. S. J. Pharm. Biomed. Anal. 2001, 25, 795-801. (23) Jaffe, M.; Hoppe-Seyler, S. Z. Phys. Chem. 1886, 10, 391-400. (24) Fanguy, J. C.; Henry, C. S. Electrophoresis 2002, 23, 767-773.
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creatininase (315 units mg-1, from Flavobacterium species), creatine, creatinine, p-aminohippuric acid, and uric acid were obtained from Sigma. The gold atomic absorption standard solution (1000 mg L-1) was purchased from Aldrich. All chemicals were used without further purification. A phosphate buffer (pH 9.5, 20 mM) solution served as the electrophoresis buffer. Stock solutions and subsequent dilutions were prepared daily in the electrophoresis buffer and were filtered with 0.45-µm filters (Gelman Acrodisc). Urine samples were collected from healthy volunteers (male, age 25-30 years) over a 24-h period and filtered in a similar fashion. Apparatus. The glass microchip (model MC-BF4-001, Micralyne Inc., Edmonton, Canada) consisted of a four-way injection cross, followed by a 62-mm-long (50-µm-wide) separation channel. The lengths of each arm (from the reservoir to the injection cross) were 5 mm. The original waste reservoir was cut off by Micralyne, leaving the channel outlet at the end side of the chip (see Figure 1), as desired for the end-column amperometric detection. A Plexiglas holder was fabricated for holding the separation chip and housing the detector and the reservoirs; exact details were given elsewhere.25 A short pipet tip was inserted in each of the reservoir holes to provide solution contact between the channel and the corresponding reservoir on the chip holder. The previously described thick-film amperometric detector25 was located in the waste reservoir (at the channel outlet side). It consisted of an Ag/AgCl wire reference electrode, a platinum wire counter electrode, and a gold-modified screen-printed carbon working electrode. The screen-printed detection electrode was placed opposite to the channel outlet (with a 50-µm distance between the electrode surface and the channel outlet). Platinum wires, inserted into the individual reservoirs, provided the electrical contact from the high-voltage leads to the solutions in the reservoirs. A homemade high-voltage power supply, containing multiple voltage terminals, was used for applying the selected driving voltage (between 0 and +4000 V) to a given reservoir and for switching between “injection” and “separation” modes. The screen-printed working electrodes were fabricated with a semiautomatic printer (model TF 100, MPM, Franklin, MA). The Acheson carbon ink Electrodag 440B (Acheson Colloids, Ontario, CA) was used for printing electrode strips. Details of the printing (25) Wang, J.; Tian, B.; Sahlin, E. Anal. Chem. 1999, 71, 5436-5440.
processes were described previously.25 The detector strip (0.4 in. × 1.333 in.) contained the working electrode (with a silver contact) printed on a ceramic substrate. The working electrode had an active area of 0.30 × 2.50 mm, as was defined by a layer of insulator. The thick-film carbon electrode was coated with a gold film, prepared by applying a square-wave pulse potential waveform (between -0.2 and 0.75 V, vs Ag/AgCl, with pulse width of 0.6 s) for 20 min in a solution containing 300 ppm Au(III), 0.1 M NaCl, and 1.5% (w/v) HCl. Amperometric detection was performed with an electrochemical analyzer 621 (CH Instruments, Austin, TX) connected to a Pentium 166-MHz computer. The electropherograms were recorded with a time resolution of 0.1 s (usually with a 7-point leastsquare smoothing) while applying the detection potential (usually +1.0 V vs Ag/AgCl wire). Sample injections were performed after baseline stabilization. All bioassays were carried out at room temperature. Electrophoresis Procedure. The channels were treated before use by rinsing with 0.1 M sodium hydroxide and deionized water for 20 and 5 min, respectively. For creatine determination (along with uric and p-aminohippuric acids), a buffer reservoir with SOx and CI was used as running buffer whereas the determination of creatinine involved the use of a buffer reservoir with SOx, CI, and CA (Figure 1). The “sample” reservoir was filled with mixtures containing creatine or creatinine (or both), paminohippuric acid, and uric acid. The detection/waste reservoir was filled with the phosphate buffer solution. The actual assays were performed by injecting the sample “plug” into the separation/reaction channel, by applying +2000 V to the sample reservoir for 3 s (with the detection reservoir grounded and other reservoirs floating). Subsequently, for simultaneous measurements of creatine or creatinine, with p-aminohippuric and uric acids, the separation voltage was applied to a running buffer reservoir containing either SOx + CI or SOx + CI + CA. Creatine or creatinine (in the sample “plug”) was thus mixed with the enzyme mixture (in the running buffer) at the channel intersection. The enzymatic reaction proceeded further down in the separation channel producing a hydrogen peroxide species. The neutral peroxide product and the p-aminohippuric and uric acids were separated in the separation/reaction channel, and the three oxidizable species were detected amperometrically at different migration times. Similar lengths of the arms coupled with proper control of the injection time ensured similar injection volumes for the subsequent analysis. The creatinine level was determined by comparing the response with and without the creatininase enzyme. For this purpose, a total (creatinine and creatine) signal was measured with the running buffer containing all three enzymes, while the creatine signal alone was recorded by applying the separation voltage to the CI-SOx reservoir. The difference in signals was used for quantifying the creatinine concentration in the sample mixture. Safety Considerations: The high-voltage power supply should be handled with extreme care to avoid electrical shock. RESULTS AND DISCUSSION In the present single-channel chip layout (Figure 1), the sample mixture and enzyme-reagent streams are mixed at the channel intersection using electrokinetic flow. The enzymatically liberated
Figure 2. Electrophoregrams for 2.5 × 10-4 M creatine (A); 2.5 × 10-4 M creatine and 2.5 × 10-4 M creatinine (B); 2.5 × 10-4 M creatine, 2.5 × 10-4 M creatinine, and 2.0 × 10-4 M p-aminohippuric acid (C); 2.5 × 10-4 M creatine, 2.5 × 10-4 M creatinine, 2.0 × 10-4 M p-aminohippuric acid; and 5.0 × 10-4 M uric acid (D). Running buffer, a phosphate buffer (20 mM, pH 9.5) solution, containing a mixture of 15, 20, and 30 units mL-1 SOx, CI, and CA, respectively (B - D) and a mixture of 15 and 20 units mL-1 SOx and CI, respectively (A). Separation potential, +1500 V; injection potential, +2000 V; injection time, 3 s; detection potential, + 1.0 V (vs Ag/ AgCl wire).
neutral peroxide species is separated from the anionic uric and p-aminohippuric acids in the separation/reaction channel, and the three oxidizable species are detected at the downstream amperometric detector. Electropherograms were recorded first to identify the individual peaks of the new multianalyte renal markers biochip (Figure 2). An injection of a 2.5 × 10-4 M creatine solution, coupled to on-column CI-SOx enzymatic reactions (eqs 2 and 3), resulted in a well-defined peak (around 110 s), associated with the oxidation of the hydrogen peroxide end product (A, a). A similar injection of a creatine/creatinine mixture in the presence of the three enzymes (CA-CI-SOx) resulted in a larger peroxide peak (B). The creatinine level in such mixture can be readily obtained from the difference in the response with and without the CA enzyme (B - A). Electropherograms C and D (of Figure 2) display the response obtained upon adding p-aminohippuric acid and uric acid, respectively, to the creatinine/creatine mixture. Such injections resulted in new well-defined peaks at 260 (b) and 335 s (c), corresponding to the oxidation of these late-migrating anionic compounds. Note that uric acid is a common interference in conventional amperometric biosensors for creatine/creatinine.12 The data of Figure 2 indicate convenient and rapid separation and detection of the different renal markers, with a total time of ∼5.5 min using a separation potential of +1500 V. Creatinine/creatine alone can actually be detected within less than 2 min. The microsystem offers effective isolation of the detector from the high separation potential, as indicated from the flat baseline and low noise level. The successful operation of the new enzyme/CE microchip requires proper attention to the levels of enzymes used. Figure 3 examines the effect of the level of sarcosine oxidase (A), creatinase (B), and creatininase (C) in the “reagent solution” upon the response to the creatine (A, B) and creatinine (C) substrates. Analytical Chemistry, Vol. 75, No. 3, February 1, 2003
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Figure 3. Effect of the SOx (A), CI (B), and CA (C) level on the response of 2.5 × 10-4 M creatine (A, B) and 5.0 × 10-4 M creatinine (C). Other conditions, as in Figure 2.
Figure 4. Hydrodynamic voltammograms for a mixture containing 2.5 × 10-4 M creatine (A), 5.0 × 10-4 M p-aminohippuric acid (B), and 1.0 × 10-3 M uric acid (C). Running buffer is a mixture of 15, 20, and 40 units mL-1 SOx, CI, and CA, respectively. Other conditions, as in Figure 2.
Similar profiles are observed for the three enzymes, with the current increasing rapidly at first upon raising the enzyme concentration and reaching a plateau thereafter. Most favorable signal-to-noise characteristics were observed with 15 units mL-1 SOx, 20 units mL-1 CI, and 30 units mL-1 CA. Higher enzyme levels resulted in an increased background noise. Hydrodynamic voltammograms (HDV) were constructed for assessing the effect of the detector potential. Figure 4 shows such HDV for uric acid (A), creatine (B), and p-aminohippuric acid (C) obtained by changing the potential of the gold-coated thick-film electrode detector over the +0.1-+1.2-V range. All compounds display well-defined characteristic sigmoidal profiles, with a current starting at +0.4 (A), +0.5 (B), and +0.8 V (C) and a leveling off above +0.7, +0.8, and +1.0 V, respectively. Subsequent work employed a potential of +1.0 V that offered the most favorable signal-to-noise characteristics. The effect of the separation voltage was examined over the 1500-4000-V range. As expected, such increase in the potential resulted in decreased migration times (e.g., from 335 to 150 s for uric acid, not shown; conditions, as in Figure 2D). However, the faster assays observed at high voltages were coupled to smaller creatinine signals, associated with shorter reaction times. The creatinine peak actually disappeared above 3000 V. Most subsequent work employed a separation voltage of 1500 V. The microchip CE system displays a well-defined concentration dependence. Figure 5 shows electrophoretic peaks for increasing levels of uric acid (A) in 1 × 10-4 M steps (b-f; 2) in the presence 528 Analytical Chemistry, Vol. 75, No. 3, February 1, 2003
Figure 5. Calibration data for uric acid (A) and creatine (B). (A): successive additions of 1 × 10-4 M uric acid (b-f) to a solution containing 1.5 × 10-4 M creatinine and creatine (a); (B) successive additions of 5.0 × 10-5 M creatine (b-f) to a solution containing 5.0 × 10-4 M uric acid (a). Other conditions, as in Figure 2A (for A) and 2D (for B).
of 1.5 × 10-4 M creatine and creatinine (1), as well as for (B) successive standard additions of 5 × 10-5 M creatine (b-f; 1) in the presence of 2.5 × 10-4 M uric acid (2). Defined peaks, proportional to the analyte concentration, are observed. The changes in concentration of one renal marker have no effect upon the response of the second one. The resulting calibration plots (not shown) were highly linear (correlation coefficients, 0.999), with sensitivities of 21.1 (creatine) and 10.0 (uric acid) nA/mM. Detection limits of 4 × 10-5 M uric acid and 2 × 10-5 M creatine can be estimated from the signal-to-noise characteristics (S/N ) 3) of peaks 2 and 1, respectively, in electropherograms b. The calibration plot for p-aminohippuric acid was also constructed, in the presence of 2.5 × 10-4 M creatinine, over a concentration range 2.0 × 10-5-1.0 × 10-4 M (not shown); this resulted in a sensitivity of 22.5 nA/mM (correlation coefficient, 0.991) and a detection limit of 2.5 × 10-5 M. The precision was evaluated from a series of eight repetitive injections of a sample mixture containing 1 × 10-4 M uric acid, 1 × 10-4 M p-aminohippuric acid, and 5 × 10-4 M creatinine mixture. All three compounds yielded reproducible peaks, with relative standard deviations of 6.6 (uric acid), 3.2 (p-aminohippuric acid), and 6.5% (creatinine) (not shown; conditions, as in Figure 2). A slight drift in the baseline and a small (5%) change in migration times were observed over prolonged operations; hence, the microchannel was washed (with 0.1 M NaOH and water) between groups of runs. The suitability of the CE microchip for measuring renal markers in clinical samples is demonstrated in Figure 6. The electropherogram for the diluted urine sample is characterized with two defined and resolved creatinine (a) and uric acid (b) peaks. No creatine peak was observed by using a “creatininasefree” two-enzyme (CI-SOx) run buffer (not shown). This also indicates the absence of neutral electroactive interferences, e.g., acetaminophen, that would otherwise have comigrated with the peroxide product. Other potential electroactive interferences, e.g., anionic ones, were not found in the urine sample tested; ascorbic acid is expected to migrate faster than uric acid.9 The total assay time is ∼340 s. Also shown in Figure 6 are electropherograms for standard additions of 2.5 × 10-4 M creatinine and 1 × 10-4 M uric acid (B, C). As expected, these additions resulted in larger creatinine and uric acid peaks and in linear standard addition plots
Figure 6. Assay of renal markers in urine sample. (A) Response for the 50-fold diluted urine sample; (B) addition of 2.5 × 10-4 M creatinine (a) and 1 × 10-4 M uric acid (b); (C) addition of 2.5 × 10-4 M creatinine (a) and 1 × 10-4 M uric acid (b). Also shown (inset) is the resulting standard additions plot. Data presented without software smoothing. Other conditions, as in Figure 2D.
(shown in the inset). The estimated levels of creatinine (6.4 × 10-3 M) and uric acid (4.0 × 10-3 M) in the original sample are consistent with the typical urine levels of these compounds.26,27 The creatinine and uric acid peaks were also changed linearly with the urine dilution factor (not shown). However, p-aminohip(26) Papa, E.; Kubota, Y.; Try, D. A.; Fujishima, A. Anal. Chem. 2000, 72, 17241727. (27) Gatti, R.; Lazzarotto, V.; De Palo, C. B.; Cappellin, E.; Spinella, P.; De Palo, E. F. Electrophoresis 1999, 20, 2917-2921. (28) Wang, J.; Pumera, M. Anal. Chem. 2002, 74, 5919-5923.
puric acid was not detected in the urine sample although the method is sensitive for its quantitation (sensitivity of 22.5 nA/ mM). Similar observations were reported elsewhere in connection with conventional CE urine analysis.22 The data of Figure 6, along with the minimal sample preparation (dilution/filtration), indicate promise for assays of physiological fluids. In conclusion, we have demonstrated a micromachined CE chip for the simultaneous measurements of several renal markers. The versatility accrued from such coupling of on-chip enzymatic assays, electrophoretic separations, and amperometric detection offers great promise for decentralized testing of renal markers. Additional sample manipulations (e.g., filtration) may be added to the chip platform, as needed to address the complexity of biological fluids. The inherent miniaturization of the electrochemical detection system makes the microscale analytical system particularly attractive for decentralized clinical testing. A dual (amperometric/conductivity) electrochemical detector28 should allow the simultaneous monitoring of additional analytes (e.g., urea, a renal clearance test marker for blood urea nitrogen, in the presence of urease). Such enzyme-based “lab-on-chip” devices would thus allow testing of renal markers to be performed more rapidly, easily, and economically in the point-of-care setting. While the concept is presented within the framework of rapid renal testing, it should eventually lead to complete high-throughput clinical microanalyzers based on parallel channels and multiple runs on a single-chip platform. ACKNOWLEDGMENT This project was supported by the National Institute of Health (NIH Grant RO1 RR14173-0). Received for review September 10, 2002. Accepted November 6, 2002. AC020560B
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