- Email: [email protected]
Multicommutated flow analysis system for determination of creatinine in physiological fluids by Jaffe method
Analytica Chimica Acta 787 (2013) 118–125
Contents lists available at SciVerse ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Multicommutated flow analysis system for determination of creatinine in physiological fluids by Jaffe method Łukasz Tymecki ∗ , Jakub Korszun, Kamil Strzelak, Robert Koncki University of Warsaw, Department of Chemistry, Pasteura 1, 02-093 Warsaw, Poland
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• Optoelectronic photometric device for Jaffe method has been developed.
• The detector has been adapted for measurements under flow analysis conditions. • The multicommuted flow system is useful for analysis of human serum and urine.
a r t i c l e
i n f o
Article history: Received 4 March 2013 Received in revised form 22 May 2013 Accepted 30 May 2013 Available online 8 June 2013 Keywords: Flow analysis Paired emitter detector diode Creatinine Jaffe method Human urine and serum analysis Clinical diagnostics
a b s t r a c t For the needs of photometric determination of creatinine according to Jaffe protocol a dedicated paired emitter detector diode (PEDD) detector has been developed. This PEDD device has been constructed in the compact form of flow-through cell (30 L total volume and 7 mm optical pathlength) integrated with 505 nm LED-based emitter and 525 nm LED-based detector compatible with multicommutated flow analysis (MCFA) system. This fully mechanized MCFA system configured of microsolenoid valves and pumps is operating under microprocessor control. The developed analytical system offers determination of creatinine in the submillimolar range of concentrations with detection limit at ppm level. The throughput offered by the system operating according to multi-point fixed-time procedure for kinetic measurements is 15–40 samples per hour depending on the mode of measurements. The developed PEDD-based MCFA system has been successfully applied for the determination of creatinine in real samples of human urine as well as serum. The developed sampling unit used the system is free from effects caused by differences in sample viscosity. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Creatinine is produced in the body by dehydration of creatine as well as by dephosphorylation of phosphocreatine. Creatine is produced in liver from glicyne, arginine, and methionine and in the phosphorylated form serves as source of energy for muscles. This way, creatinine is a marker for muscle mass and its generation reflects slow turnover of muscle protein. The creatinine level may be regarded as an index of amino acid pool stored in the muscle mass and any reduction in this index over time means propor-
∗ Corresponding author. Tel.: +48 22 8220211. E-mail address: [email protected] (Ł. Tymecki). 0003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2013.05.052
tional loss of nitrogen reserve. Moreover, creatinine is recognized as a low molecular weight model uremic toxin. Apart from glucose, Creatinine is the most requested analyte in modern clinical analysis [1]. Determination of this metabolite in various physiological fluids is useful for the evaluation of muscular, renal and thyroid dysfunctions. Such analyses are helpful for the biomedical diagnosis of acute myocardial infarction as well as for the description of kidney diseases. Physiological creatinine level in human serum varies from around 0.05–0.11 mM. Creatinine serum concentrations higher than 0.14 mM require further clinical investigations and those over 0.50 mM indicate severe renal dysfunction. In extremely pathological conditions the concentration can exceed 1.0 mM. On the other hand, significantly higher levels of creatinine (several millimoles per liter) are found in urine samples from healthy people. Urinary
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Fig. 1. Changes of solution spectrum in the course of Jaffe reaction. Additionally, the emission spectra of LEDs tested as PEDD components are shown (A). The scheme of measurement setup (B) and the response for 1.0, 0.5, 025 and 0.125 mM creatinine for each LED-LED combination (C).
creatinine decreases in muscular atrophy and myotonic dystrophy thus it can be applied as a quantitative indicator of anorexia. Max Jaffe is known for having given his name to an analytical principle for assaying creatinine in human body fluids. According to the historical note [2], chronologically the substance creatinine was first named by Liebig (1847) and synthesized by Horbaczewski (1885). Jaffe (1886) described its reaction with alkaline picrate, however the analytical utility of this reaction for creatinine assaying in urine (1904) and blood (1914) was demonstrated by Folin. Over hundred years following the discovery of the principle, the Jaffe method still remains dominant in clinical laboratories [3,4], which is a fact unparalleled in clinical chemistry. Despite the advent of enzymatic and separation methods for creatinine analysis, the Jaffe method is still popular due to its simplicity and low cost. Owing to the requirements of routine clinical analysis the kinetic protocol of Jaffe method often is the subject of automation. Several flow analysis systems dedicated for photometric determination of creatinine in the form of color complex with picrate are reported in the literature [5–14]. Some of them are realized in the sequential injection analysis [11,12] as well as microfluidic format [13,14]. In almost all cited papers for photometric detection of creatinine–picrate complex conventional spectrophotometers with flow-through cuvettes or fiber optic spectrophotometers are applied, although there is no need of measurements at several wavelengths. In this paper we present optoelectronic detector dedicated for Jaffe method constructed according to paired emitter detector diode (PEDD) principle [15–17]. As demonstrated elsewhere, due to high dynamics of response PEDD-based detectors are useful for kinetic measurements under non-stationary conditions [18–20] as well as for monitoring of reactions directly inside flow-through reaction microchambers [21,22] and open-tubular bioreactors [23]. In this paper a low-cost, fibreless and compact PEDD-based photometric device is applied in the multicommutated flow analysis (MCFA) system based on microprocessor-controlled microsolenoid pumps and valves. The utility of developed
PEDD-MCFA system for analysis of real samples of clinical significance will be tested. 2. Experimental Creatinine (as hydrochloride) has been obtained from Sigma–Aldrich (USA). Other chemicals of analytical grade were obtained from POCh (Poland). Doubly distilled water was used throughout. Serum standards with physiological and pathological creatinine content were obtained from Cormay (Poland). These lyophilized control sera were reconstituted with water two hours before use and stored at room temperature no longer than 12 h. Real samples of human urine and serum with known creatinine levels applied for the validation of developed analytical system were obtained from Central Military Hospital in Warsaw. The reference analyses of physiological fluids were performed according to clinically recommended Jaffe protocol [3,4] in the clinical laboratory using commercial clinical analyzer Cobas Integra 600 system (Roche Diagnostics, USA). The LEDs (5 mm diameter, transparent lens, flat front; 40◦ view angle) for PEDD development were obtained from Optosupply (Hong Kong). Electromotive force generated by PEDD, treated as an analytical signal [15,16], was measured and recorded with UNIT multimeter (model UT70B, China) operating in the voltmeter mode and connected with data storage PC via RS232 interface. Microsolenoid pumps (operating with 2 Hz frequency and indicated stroke volume of 10 L, product no. 120SP1210-4TE) and threeway microsolenoid valves (product no. 100T3MP12-62-5) applied for the construction of MCFA system were purchased from BioChem Fluidics (Boonton, USA). These devices and resulting MCFA system were microprocessor-controlled using the KSP Measuring System (Poland) [23,24]. Flow analysis manifold was arranged using PTFE Microbore tubing (ID of 0.8 mm) from Cole-Palmer (USA).
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Fig. 2. Kinetic measurements performed in the cuvette format with PEDD powering by different currents (top) and corresponding calibration graphs obtained for different reaction times according to single-point fixed-time mode of measurements (bottom).
3. Results and discussion 3.1. PEDD detector for Jaffe method In the course of Jaffe reaction, creatinine reacts directly with picrate under alkaline conditions to form equimolar complex which red-orange color is detected and quantified. Fig. 1A presents the changes of spectrum associated with the color development in the course of reaction progress registered every 30 s. Additionally, the emission spectra of 505 nm and 525 nm LEDs are shown. The first goal of experiments was the selection of appropriate LEDs for PEDD construction. The measurements were performed in simple experimental setup depicted in Fig. 1B. The tested LEDs were mounted into cuvette holder made of Lego bricks (shown elsewhere [15,17]). The holder fits to standard cuvettes with 1 cm of optical pathlength and common 5 mm diameter LEDs. All four combination of paired LEDs has been tested and the resulting time courses for creatinine–picrate formation are shown in Fig. 1C. The kinetic measurements were performed for 1.00, 0.50, 0.25 and 0.125 mM creatinine in the presence of picric acid (0.025 mM) and NaOH (0.2 mM). The baseline signals were obtained for yellow blank solution containing picrates in alkaline solution but no target analyte. Evidently, the most promising PEDD for Jaffe method consists of 505 nm LED-emitter and 525 nm LED-detector. As expected, the worst is the opposite LED-LED configuration. The results shown in the Fig. 1C stay in agreement with earlier observations [15] that LED operating in the reverse mode (as a light
detector) exhibits the highest sensitivity for the light of slightly higher energy than it is able to emit. The PEDD offering the highest sensitivity has been applied for further investigations. Fig. 2 (top) shows the results of kinetic measurements performed at different PEDD powering currents. The increase of current causes the increase of assay sensitivity and such observation is in agreement with earlier predictions [16]. To avoid the burnout of LED-emitter the experiments at currents higher that those recommended by the LED manufacturer were not performed and therefore a common effect of the shift of calibration graph toward higher concentrations [16] was not observed. The presented readouts are useful for various modes of kinetic measurements. The corresponding calibration graphs shown in Fig. 2 (bottom) have been obtained according to single-point fixed-time method, where the signal is measured over a predetermined time interval. As expected, the increase of time of assay results in the increase of sensitivity but for higher concentrations the loss of wide-range linear response of PEDD is observed. This upper determination limit is caused by the total discharging of weakly illuminated LED-detector [15,16]. Taking into account the data shown in Fig. 2 it can be summarized that the manual cuvette assay performed with the developed PEDD dedicated for Jaffe method allows the determination of creatinine in the submillimolar range of concentrations with the detection limit below 1 ppm within a few minutes only. The time-dependent sensitivity of this assay in the linear range of response varies from 180 mV mM−1 to 1648 mV mM−1 . LEDs can be integrated in the form of flow-through PEDD by simple gluing of their bulbs bodies with beforehand milling
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Fig. 3. MCFA manifold for testing of flow-through PEDD detector for Jaffe method (top), the calibration of PEDD-MCFA system (middle) and corresponding calibration graphs (bottom) obtained at different currents supplying the detector according to single-point fixed-time mode of measurements. The interval times (in seconds) are given in the figure.
flow channels [15]. However, in this study the device with easily replaced LEDs mounted into the dedicated flow-cell made of PEEK has been applied. The cross-section of such device is given as a supplementary material (Fig. S1) to this article. The internal volume and the optical pathlength of applied flow-cell are 30 L and 7 mm, respectively. The effects of internal geometry of photometric flow-through PEDDs on their analytical features have been investigated in detail and presented elsewhere [20]. The developed device was primary tested in a simple flow analysis manifold constructed of microsolenoids shown in Fig. 3 (top). This system contains pumpvalve module enabling the calibration of flow-through detector using only one, single analyte standard [25]. The operation of valve V1 defines standard dilution ratio, while the pump provides the homogenization of reaction segment. The concentration of creatinine standard was 0.10 mM. The concentrations of applied picric acid and NaOH solutions were 0.025 mM and 0.2 mM, respectively.
Fig. 3 (middle) presents the calibrations of detector in the flow system performed at different currents applied for PEDD supplying. The volume of creatinine standard used for single peak generation was 0.8 mL or proportionally less according to required dilution ratio [25]. The generated reaction segment is stopped inside PEDD detector and the changes of its absorbance are monitored. At the bottom of Fig. 3 the corresponding calibration graphs are shown. The graphs were constructed according to one-point fixedtime methodology of kinetic measurements, where as an analytical signal the difference between electromotive force of PEDD recorded in the absence and presence of analyte was used. The obtained calibration graphs are only slightly current-dependent, although the baseline for water as well as the signals for Jaffe reagents without target analyte significantly shift with the current. This observation confirms, that the changes of absorbance connected with Jaffe reaction are monitored within the range of linear response of PEDD
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Fig. 4. Improved MCFA manifold (top) and the recording of calibration cycle (bottom). In the inset the two stages of sampling delivery unit (filling and injection) are depicted.
detector. It implies, that the errors caused by possible fluctuations of LED-emitter will be minimized. The results shown in Fig. 3 (bottom) clearly indicate that the flow-through PEDD exhibits linear response to creatinine enabling its determination in the micromolar range of concentrations with sensitivity strongly dependent on time of assay (from 492 mV mM−1 for 30 s to 2590 mV mM−1 for 180 s). Obviously, the increase of reaction time causes the decrease of injection frequency, thus the proper selection of this time results from the compromise between required sensitivity and throughput of the analytical system. 3.2. PEDD-MCFA system for analysis of physiological fluids The analytical parameters offered by MCFA system shown in Fig. 3 are absolutely sufficient for urine analysis. Taking into account the range of determination offered by the system (Fig. 4) the urine samples should be 100-fold diluted and this way the non-specific effects from viscosity and color of urine are efficiently eliminated. Unfortunately, in case of serum analysis the MCFA system totally fails generating highly irreproducible peaks. Main reason of this drawback is the viscosity of serum. The pump located at the end
of the flow system is not able to aspirate reproducible volumes of segments of different viscosity. Moreover, the operation of the MCFA manifold requires relatively high amount of sample, whilst, contrary to urine, serum is rather limited sample. To solve such problems the MCFA system has to be reconfigured. The improved MCFA manifold containing sample delivery cell (SDC) and additional pump is shown in Fig. 4 (top). The cross-section of SDC is given as a supplementary material (Fig. S2) to this article. The internal volume of this device (i.e. volume of sampling segment) is 8 L. The operation of SDC is depicted in the inset of Fig. 4. The valve V3 together with the pump P2 forms the module for filling the SDC which plays similar role as a sample loop in conventional injection valve. The pump P2 aspired the sample through the valve V2 with mounted stopper (operating as a check valve). At this time the flow in the main line (to detector as well as to containers with reagents) is closed by the pump P1 and the valve V3 with stopper. In the second SDC stage (injection, see inset of Fig. 4) the sample segment is forced by pump P1 into PEDD detector, whereas the sampling line is closed by valve V2 and pump P2. The role of valve V1 (like in the former flow manifold) is the mixing of reagents required for the assay.
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Fig. 5. Calibration graphs, obtained from the recording shown in Fig. 4, dependent on applied delay and interval times (shown in Figure insets) of two-point fixed-time kinetic procedure.
In the improved MCFA system the reagents (including picrate) are permanently present in the PEDD detector and therefore the baseline signal for colorless solution is not registered. A recording of a common calibration is shown in the bottom of Fig. 4. The “peaks” are useful for direct construction of calibration curve according to single-point method, however the multi-point fixed-time methodology is also possible. As shown in Fig. 5, the choice of delay and interval times results in significantly different calibration graphs in respect to offered sensitivity and range of linear response. For higher intervals a higher sensitivity is obtained. On the other hand higher delay times the bigger deviations from linearity of response are observed. However, in all cases the reported PEDD-MCFA system offers linear responses for creatinine up to 250 M. In the linear range of response the sensitivity for 45, 90, 135 and 180 s intervals is 440, 844, 1160 and 1464 mV mM−1 , respectively. As can be seen from Fig. 5, the reproducibility of measurements is satisfactory (see error bars at each calibration point). Obviously, the highest sensitivities and widest ranges of linear response are obtained when delay time is equal zero, i.e. for data processing according to one-point fixed-time methodology (Fig. 5). However, in case of real samples analysis the approach based on two-point fixed time methodology can be useful for elimination of interferences from color matrix as well as from some non-creatinine reactants interfering as alternative chromogens in Jaffe method [4,9]. In Fig. 6 analytical signals recorded for diluted urine (top) and serum (bottom) sample are shown. In each case the signals are well reproducible (SD = 2.9 mV for urine and SD = 1.7 mV for serum, RSD < 1% in both cases), however the effects from serum matrix are significant especially for shorter times. The final goal of these investigations was the analysis of physiological fluids. The developed PEDD-based MCFA system was validated using samples of human urine and serum previously analyzed in clinical laboratory settings applying recommended method and commercial analyzer. 30 samples of urine and 27 samples of serum were analyzed. Before injection urine samples
were diluted. The serum samples exhibiting extremely high, creatinine levels (over 300 M) were diluted twice before analysis. As it can be seen from Fig. 7 (right), the results of human urine and serum analysis are fully compatible with those obtained using the routine methodology. For elaboration of measurement data from PEDD-MCFA system two-point fixed-time method was examined. The influence of delay/interval times on the values of correlation parameters (coefficient, slope and intercept) are shown in the left part of Fig. 7.
Fig. 6. Signals recorded in the MCFA system for 100-fold diluted urine (top) and undiluted serum (bottom) samples. Data for triplicate injection.
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Fig. 7. Results of urine (A) and serum analysis using water standards (B) and control sera (C) as calibrants. The correlation between the developed and reference method (right) and the dependences of correlation parameters (coefficient, slope and intercept) on delay/interval time (left). Point sizes indicate interval – from 45 s (smallest points) to 180 s (largest points).
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As expected, in case of urine analysis the regression coefficients between developed and routine methods are almost independent of delay/interval time (and vary from 0.981 to 0.993). This finding is not surprised, because diluted samples of urine are practically colorless (see Fig. 6). In case of undiluted serum analysis the results obtained without delaying (delay time = 0, one-point methodology) are significantly different from these where the elimination of matrix effect by two-point fixed-time methodology of measurements is applied. This difference is observed also in case of serum analysis performed with control sera used as standards for calibration. The best regression coefficients (obtained for 150 s delay and 45 s interval) for analysis performed with artificial water standards and control sera as calibrants were, 0.980 and 0.979, respectively. It can be concluded, that the methodology of data processing is predominant and stronger affects the final results of analysis than the choice of standards for calibration. 4. Conclusions Although optical detection dominates in analytical chemistry for many real applications highly advanced and expensive spectrophotometers are not necessary. For many particular analytical uses simple optical detectors based on extremely cheap optoelectronic components are absolutely sufficient. This statement is especially valid in case of flow analysis, where the systems are developed with the assumption that they are dedicated for particular kind of target analyte, method and sample. In this contribution an extremely cheap photometric PEDD-based detector for Jaffe method operating with low-power source and cheap instrumentation (voltmeter) has been developed and validated. Its utility for real analytics has been confirmed by its implementation into MCFA system and successful application for human urine and serum analysis. It should be pointed out that microsolenoid devices applied in the developed MCFA system also belong to the group of low-budget components useful for construction of flow analysis manifolds. In conclusion, it is worth to indicate further potential biomedical applications of the developed PEDD detector. One of the current trends in clinical urinalysis [26] is the diagnostics of microproteinuria as well as microalbuminuria based on the estimation of creatinine to protein ratio [12,14]. The PEDD concept presented in this paper can be useful for such diagnostics, especially that only recently photometric and fluorimetric [27] as well as turbidimetric and nephelometric [28] PEDD-based detectors dedicated for protein determination have been developed. Another area important from the biomedical point of view are monitors of creatinine removal by artificial kidneys, as this metabolite is established as a model uremic toxin useful for quantitative description of
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hemodialysis efficiency and adequacy [29]. The results presented in this paper evidenced that the determination range offered by the developed PEDD-based detector and MCFA system are also sufficient for such kind of biomedical applications. Acknowledgement These investigations were granted by the Polish National Science Centre (Project Opus NCN no. 2011/01/B/NZ5/00934). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2013.05.052. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29]
K. Spencer, Ann. Clin. Chem. 23 (1986) 1–25. J.R. Delanghe, M.M. Speeckaert, Nephrol. Dial. Transpl. 4 (2011) 83–86. J. Vasiliades, Clin. Chem. 22 (1976) 1664–1671. L.D. Bowers, E.T. Wong, Clin. Chem. 26 (1980) 555–561. J.F. van Staden, Z. Fresenius, Anal. Chem. 315 (1983) 141–144. G. del Campo, A. Irastorza, J.A. Casado, Fresenius J. Anal. Chem. 352 (1995) 557–561. T. Sakai, H. Ohta, N. Ohno, J. Imai, Anal. Chim. Acta 308 (1995) 446–450. A.N. Araujo, J.L.F. Costa Lima, B.F. Reis, E.A.G. Zagatto, Anal. Chim. Acta 310 (1995) 447–452. P. Campins Falco, L.A. Tortajada Genaro, S. Meseger Lloret, F. Blasco Gomez, A. Sevillano Cabeza, C. Molins Legua, Talanta 55 (2001) 1079–1089. I. Grabowska, M. Chudy, A. Dybko, Z. Brzozka, Anal. Chim. Acta 540 (2005) 181–185. S.I. Ohira, A.B. Kirk, P.K. Dasgupta, Anal. Biochem. 384 (2009) 238–244. W. Siangproh, N. Teshima, T. Sakai, S. Katoh, O. Chailapakul, Talanta 79 (2009) 1111–1117. T. Songjaroen, T. Maturos, A. Sappat, A. Tuantranont, W. Laiwattanapaisal, Anal. Chim. Acta 647 (2009) 78–83. C.C. Lin, J.L. Hsu, C.C. Tseng, G.B. Lee, Microfluid. Nanofluid. 10 (2011) 1055–1067. L. Tymecki, M. Pokrzywnicka, R. Koncki, Analyst 133 (2008) 1501–1504. L. Tymecki, R. Koncki, Anal. Chim. Acta 639 (2009) 73–77. M. Pokrzywnicka, R. Koncki, L. Tymecki, Chem. Anal. 54 (2009) 427–435. L. Tymecki, L. Brodacka, B. Rozum, R. Koncki, Analyst 134 (2009) 1333–1337. K. Strzelak, R. Koncki, L. Tymecki, Talanta 96 (2012) 127–131. L. Tymecki, K. Strzelak, R. Koncki, Anal. Chim. Acta (2013), submitted for publication. S. Koronkiewicz, S. Kalinowski, Talanta 86 (2011) 436–441. S. Koronkiewicz, S. Kalinowski, Talanta 96 (2012) 68–74. B. Rozum, K. Gajownik, L. Tymecki, R. Koncki, Anal. Biochem. 400 (2010) 151–153. http://debiany.pl/ksp/index.html L. Tymecki, K. Strzelak, R. Koncki, Talanta 79 (2009) 205–210. C.C. Lin, G.B. Lee, Analyst 136 (2011) 2669–2688. M. Pokrzywnicka, L. Tymecki, R. Koncki, Talanta 96 (2012) 121–126. K. Strzelak, R. Koncki, Anal. Chim. Acta (2013), http://dx.doi.org/10.1016/j.aca. 2013.06.003, accepted for publication. R. Koncki, Trends Anal. Chem. 27 (2008) 304–314.
Contents lists available at SciVerse ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Multicommutated flow analysis system for determination of creatinine in physiological fluids by Jaffe method Łukasz Tymecki ∗ , Jakub Korszun, Kamil Strzelak, Robert Koncki University of Warsaw, Department of Chemistry, Pasteura 1, 02-093 Warsaw, Poland
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• Optoelectronic photometric device for Jaffe method has been developed.
• The detector has been adapted for measurements under flow analysis conditions. • The multicommuted flow system is useful for analysis of human serum and urine.
a r t i c l e
i n f o
Article history: Received 4 March 2013 Received in revised form 22 May 2013 Accepted 30 May 2013 Available online 8 June 2013 Keywords: Flow analysis Paired emitter detector diode Creatinine Jaffe method Human urine and serum analysis Clinical diagnostics
a b s t r a c t For the needs of photometric determination of creatinine according to Jaffe protocol a dedicated paired emitter detector diode (PEDD) detector has been developed. This PEDD device has been constructed in the compact form of flow-through cell (30 L total volume and 7 mm optical pathlength) integrated with 505 nm LED-based emitter and 525 nm LED-based detector compatible with multicommutated flow analysis (MCFA) system. This fully mechanized MCFA system configured of microsolenoid valves and pumps is operating under microprocessor control. The developed analytical system offers determination of creatinine in the submillimolar range of concentrations with detection limit at ppm level. The throughput offered by the system operating according to multi-point fixed-time procedure for kinetic measurements is 15–40 samples per hour depending on the mode of measurements. The developed PEDD-based MCFA system has been successfully applied for the determination of creatinine in real samples of human urine as well as serum. The developed sampling unit used the system is free from effects caused by differences in sample viscosity. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Creatinine is produced in the body by dehydration of creatine as well as by dephosphorylation of phosphocreatine. Creatine is produced in liver from glicyne, arginine, and methionine and in the phosphorylated form serves as source of energy for muscles. This way, creatinine is a marker for muscle mass and its generation reflects slow turnover of muscle protein. The creatinine level may be regarded as an index of amino acid pool stored in the muscle mass and any reduction in this index over time means propor-
∗ Corresponding author. Tel.: +48 22 8220211. E-mail address: [email protected] (Ł. Tymecki). 0003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2013.05.052
tional loss of nitrogen reserve. Moreover, creatinine is recognized as a low molecular weight model uremic toxin. Apart from glucose, Creatinine is the most requested analyte in modern clinical analysis [1]. Determination of this metabolite in various physiological fluids is useful for the evaluation of muscular, renal and thyroid dysfunctions. Such analyses are helpful for the biomedical diagnosis of acute myocardial infarction as well as for the description of kidney diseases. Physiological creatinine level in human serum varies from around 0.05–0.11 mM. Creatinine serum concentrations higher than 0.14 mM require further clinical investigations and those over 0.50 mM indicate severe renal dysfunction. In extremely pathological conditions the concentration can exceed 1.0 mM. On the other hand, significantly higher levels of creatinine (several millimoles per liter) are found in urine samples from healthy people. Urinary
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Fig. 1. Changes of solution spectrum in the course of Jaffe reaction. Additionally, the emission spectra of LEDs tested as PEDD components are shown (A). The scheme of measurement setup (B) and the response for 1.0, 0.5, 025 and 0.125 mM creatinine for each LED-LED combination (C).
creatinine decreases in muscular atrophy and myotonic dystrophy thus it can be applied as a quantitative indicator of anorexia. Max Jaffe is known for having given his name to an analytical principle for assaying creatinine in human body fluids. According to the historical note [2], chronologically the substance creatinine was first named by Liebig (1847) and synthesized by Horbaczewski (1885). Jaffe (1886) described its reaction with alkaline picrate, however the analytical utility of this reaction for creatinine assaying in urine (1904) and blood (1914) was demonstrated by Folin. Over hundred years following the discovery of the principle, the Jaffe method still remains dominant in clinical laboratories [3,4], which is a fact unparalleled in clinical chemistry. Despite the advent of enzymatic and separation methods for creatinine analysis, the Jaffe method is still popular due to its simplicity and low cost. Owing to the requirements of routine clinical analysis the kinetic protocol of Jaffe method often is the subject of automation. Several flow analysis systems dedicated for photometric determination of creatinine in the form of color complex with picrate are reported in the literature [5–14]. Some of them are realized in the sequential injection analysis [11,12] as well as microfluidic format [13,14]. In almost all cited papers for photometric detection of creatinine–picrate complex conventional spectrophotometers with flow-through cuvettes or fiber optic spectrophotometers are applied, although there is no need of measurements at several wavelengths. In this paper we present optoelectronic detector dedicated for Jaffe method constructed according to paired emitter detector diode (PEDD) principle [15–17]. As demonstrated elsewhere, due to high dynamics of response PEDD-based detectors are useful for kinetic measurements under non-stationary conditions [18–20] as well as for monitoring of reactions directly inside flow-through reaction microchambers [21,22] and open-tubular bioreactors [23]. In this paper a low-cost, fibreless and compact PEDD-based photometric device is applied in the multicommutated flow analysis (MCFA) system based on microprocessor-controlled microsolenoid pumps and valves. The utility of developed
PEDD-MCFA system for analysis of real samples of clinical significance will be tested. 2. Experimental Creatinine (as hydrochloride) has been obtained from Sigma–Aldrich (USA). Other chemicals of analytical grade were obtained from POCh (Poland). Doubly distilled water was used throughout. Serum standards with physiological and pathological creatinine content were obtained from Cormay (Poland). These lyophilized control sera were reconstituted with water two hours before use and stored at room temperature no longer than 12 h. Real samples of human urine and serum with known creatinine levels applied for the validation of developed analytical system were obtained from Central Military Hospital in Warsaw. The reference analyses of physiological fluids were performed according to clinically recommended Jaffe protocol [3,4] in the clinical laboratory using commercial clinical analyzer Cobas Integra 600 system (Roche Diagnostics, USA). The LEDs (5 mm diameter, transparent lens, flat front; 40◦ view angle) for PEDD development were obtained from Optosupply (Hong Kong). Electromotive force generated by PEDD, treated as an analytical signal [15,16], was measured and recorded with UNIT multimeter (model UT70B, China) operating in the voltmeter mode and connected with data storage PC via RS232 interface. Microsolenoid pumps (operating with 2 Hz frequency and indicated stroke volume of 10 L, product no. 120SP1210-4TE) and threeway microsolenoid valves (product no. 100T3MP12-62-5) applied for the construction of MCFA system were purchased from BioChem Fluidics (Boonton, USA). These devices and resulting MCFA system were microprocessor-controlled using the KSP Measuring System (Poland) [23,24]. Flow analysis manifold was arranged using PTFE Microbore tubing (ID of 0.8 mm) from Cole-Palmer (USA).
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Fig. 2. Kinetic measurements performed in the cuvette format with PEDD powering by different currents (top) and corresponding calibration graphs obtained for different reaction times according to single-point fixed-time mode of measurements (bottom).
3. Results and discussion 3.1. PEDD detector for Jaffe method In the course of Jaffe reaction, creatinine reacts directly with picrate under alkaline conditions to form equimolar complex which red-orange color is detected and quantified. Fig. 1A presents the changes of spectrum associated with the color development in the course of reaction progress registered every 30 s. Additionally, the emission spectra of 505 nm and 525 nm LEDs are shown. The first goal of experiments was the selection of appropriate LEDs for PEDD construction. The measurements were performed in simple experimental setup depicted in Fig. 1B. The tested LEDs were mounted into cuvette holder made of Lego bricks (shown elsewhere [15,17]). The holder fits to standard cuvettes with 1 cm of optical pathlength and common 5 mm diameter LEDs. All four combination of paired LEDs has been tested and the resulting time courses for creatinine–picrate formation are shown in Fig. 1C. The kinetic measurements were performed for 1.00, 0.50, 0.25 and 0.125 mM creatinine in the presence of picric acid (0.025 mM) and NaOH (0.2 mM). The baseline signals were obtained for yellow blank solution containing picrates in alkaline solution but no target analyte. Evidently, the most promising PEDD for Jaffe method consists of 505 nm LED-emitter and 525 nm LED-detector. As expected, the worst is the opposite LED-LED configuration. The results shown in the Fig. 1C stay in agreement with earlier observations [15] that LED operating in the reverse mode (as a light
detector) exhibits the highest sensitivity for the light of slightly higher energy than it is able to emit. The PEDD offering the highest sensitivity has been applied for further investigations. Fig. 2 (top) shows the results of kinetic measurements performed at different PEDD powering currents. The increase of current causes the increase of assay sensitivity and such observation is in agreement with earlier predictions [16]. To avoid the burnout of LED-emitter the experiments at currents higher that those recommended by the LED manufacturer were not performed and therefore a common effect of the shift of calibration graph toward higher concentrations [16] was not observed. The presented readouts are useful for various modes of kinetic measurements. The corresponding calibration graphs shown in Fig. 2 (bottom) have been obtained according to single-point fixed-time method, where the signal is measured over a predetermined time interval. As expected, the increase of time of assay results in the increase of sensitivity but for higher concentrations the loss of wide-range linear response of PEDD is observed. This upper determination limit is caused by the total discharging of weakly illuminated LED-detector [15,16]. Taking into account the data shown in Fig. 2 it can be summarized that the manual cuvette assay performed with the developed PEDD dedicated for Jaffe method allows the determination of creatinine in the submillimolar range of concentrations with the detection limit below 1 ppm within a few minutes only. The time-dependent sensitivity of this assay in the linear range of response varies from 180 mV mM−1 to 1648 mV mM−1 . LEDs can be integrated in the form of flow-through PEDD by simple gluing of their bulbs bodies with beforehand milling
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Fig. 3. MCFA manifold for testing of flow-through PEDD detector for Jaffe method (top), the calibration of PEDD-MCFA system (middle) and corresponding calibration graphs (bottom) obtained at different currents supplying the detector according to single-point fixed-time mode of measurements. The interval times (in seconds) are given in the figure.
flow channels [15]. However, in this study the device with easily replaced LEDs mounted into the dedicated flow-cell made of PEEK has been applied. The cross-section of such device is given as a supplementary material (Fig. S1) to this article. The internal volume and the optical pathlength of applied flow-cell are 30 L and 7 mm, respectively. The effects of internal geometry of photometric flow-through PEDDs on their analytical features have been investigated in detail and presented elsewhere [20]. The developed device was primary tested in a simple flow analysis manifold constructed of microsolenoids shown in Fig. 3 (top). This system contains pumpvalve module enabling the calibration of flow-through detector using only one, single analyte standard [25]. The operation of valve V1 defines standard dilution ratio, while the pump provides the homogenization of reaction segment. The concentration of creatinine standard was 0.10 mM. The concentrations of applied picric acid and NaOH solutions were 0.025 mM and 0.2 mM, respectively.
Fig. 3 (middle) presents the calibrations of detector in the flow system performed at different currents applied for PEDD supplying. The volume of creatinine standard used for single peak generation was 0.8 mL or proportionally less according to required dilution ratio [25]. The generated reaction segment is stopped inside PEDD detector and the changes of its absorbance are monitored. At the bottom of Fig. 3 the corresponding calibration graphs are shown. The graphs were constructed according to one-point fixedtime methodology of kinetic measurements, where as an analytical signal the difference between electromotive force of PEDD recorded in the absence and presence of analyte was used. The obtained calibration graphs are only slightly current-dependent, although the baseline for water as well as the signals for Jaffe reagents without target analyte significantly shift with the current. This observation confirms, that the changes of absorbance connected with Jaffe reaction are monitored within the range of linear response of PEDD
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Fig. 4. Improved MCFA manifold (top) and the recording of calibration cycle (bottom). In the inset the two stages of sampling delivery unit (filling and injection) are depicted.
detector. It implies, that the errors caused by possible fluctuations of LED-emitter will be minimized. The results shown in Fig. 3 (bottom) clearly indicate that the flow-through PEDD exhibits linear response to creatinine enabling its determination in the micromolar range of concentrations with sensitivity strongly dependent on time of assay (from 492 mV mM−1 for 30 s to 2590 mV mM−1 for 180 s). Obviously, the increase of reaction time causes the decrease of injection frequency, thus the proper selection of this time results from the compromise between required sensitivity and throughput of the analytical system. 3.2. PEDD-MCFA system for analysis of physiological fluids The analytical parameters offered by MCFA system shown in Fig. 3 are absolutely sufficient for urine analysis. Taking into account the range of determination offered by the system (Fig. 4) the urine samples should be 100-fold diluted and this way the non-specific effects from viscosity and color of urine are efficiently eliminated. Unfortunately, in case of serum analysis the MCFA system totally fails generating highly irreproducible peaks. Main reason of this drawback is the viscosity of serum. The pump located at the end
of the flow system is not able to aspirate reproducible volumes of segments of different viscosity. Moreover, the operation of the MCFA manifold requires relatively high amount of sample, whilst, contrary to urine, serum is rather limited sample. To solve such problems the MCFA system has to be reconfigured. The improved MCFA manifold containing sample delivery cell (SDC) and additional pump is shown in Fig. 4 (top). The cross-section of SDC is given as a supplementary material (Fig. S2) to this article. The internal volume of this device (i.e. volume of sampling segment) is 8 L. The operation of SDC is depicted in the inset of Fig. 4. The valve V3 together with the pump P2 forms the module for filling the SDC which plays similar role as a sample loop in conventional injection valve. The pump P2 aspired the sample through the valve V2 with mounted stopper (operating as a check valve). At this time the flow in the main line (to detector as well as to containers with reagents) is closed by the pump P1 and the valve V3 with stopper. In the second SDC stage (injection, see inset of Fig. 4) the sample segment is forced by pump P1 into PEDD detector, whereas the sampling line is closed by valve V2 and pump P2. The role of valve V1 (like in the former flow manifold) is the mixing of reagents required for the assay.
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Fig. 5. Calibration graphs, obtained from the recording shown in Fig. 4, dependent on applied delay and interval times (shown in Figure insets) of two-point fixed-time kinetic procedure.
In the improved MCFA system the reagents (including picrate) are permanently present in the PEDD detector and therefore the baseline signal for colorless solution is not registered. A recording of a common calibration is shown in the bottom of Fig. 4. The “peaks” are useful for direct construction of calibration curve according to single-point method, however the multi-point fixed-time methodology is also possible. As shown in Fig. 5, the choice of delay and interval times results in significantly different calibration graphs in respect to offered sensitivity and range of linear response. For higher intervals a higher sensitivity is obtained. On the other hand higher delay times the bigger deviations from linearity of response are observed. However, in all cases the reported PEDD-MCFA system offers linear responses for creatinine up to 250 M. In the linear range of response the sensitivity for 45, 90, 135 and 180 s intervals is 440, 844, 1160 and 1464 mV mM−1 , respectively. As can be seen from Fig. 5, the reproducibility of measurements is satisfactory (see error bars at each calibration point). Obviously, the highest sensitivities and widest ranges of linear response are obtained when delay time is equal zero, i.e. for data processing according to one-point fixed-time methodology (Fig. 5). However, in case of real samples analysis the approach based on two-point fixed time methodology can be useful for elimination of interferences from color matrix as well as from some non-creatinine reactants interfering as alternative chromogens in Jaffe method [4,9]. In Fig. 6 analytical signals recorded for diluted urine (top) and serum (bottom) sample are shown. In each case the signals are well reproducible (SD = 2.9 mV for urine and SD = 1.7 mV for serum, RSD < 1% in both cases), however the effects from serum matrix are significant especially for shorter times. The final goal of these investigations was the analysis of physiological fluids. The developed PEDD-based MCFA system was validated using samples of human urine and serum previously analyzed in clinical laboratory settings applying recommended method and commercial analyzer. 30 samples of urine and 27 samples of serum were analyzed. Before injection urine samples
were diluted. The serum samples exhibiting extremely high, creatinine levels (over 300 M) were diluted twice before analysis. As it can be seen from Fig. 7 (right), the results of human urine and serum analysis are fully compatible with those obtained using the routine methodology. For elaboration of measurement data from PEDD-MCFA system two-point fixed-time method was examined. The influence of delay/interval times on the values of correlation parameters (coefficient, slope and intercept) are shown in the left part of Fig. 7.
Fig. 6. Signals recorded in the MCFA system for 100-fold diluted urine (top) and undiluted serum (bottom) samples. Data for triplicate injection.
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Fig. 7. Results of urine (A) and serum analysis using water standards (B) and control sera (C) as calibrants. The correlation between the developed and reference method (right) and the dependences of correlation parameters (coefficient, slope and intercept) on delay/interval time (left). Point sizes indicate interval – from 45 s (smallest points) to 180 s (largest points).
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As expected, in case of urine analysis the regression coefficients between developed and routine methods are almost independent of delay/interval time (and vary from 0.981 to 0.993). This finding is not surprised, because diluted samples of urine are practically colorless (see Fig. 6). In case of undiluted serum analysis the results obtained without delaying (delay time = 0, one-point methodology) are significantly different from these where the elimination of matrix effect by two-point fixed-time methodology of measurements is applied. This difference is observed also in case of serum analysis performed with control sera used as standards for calibration. The best regression coefficients (obtained for 150 s delay and 45 s interval) for analysis performed with artificial water standards and control sera as calibrants were, 0.980 and 0.979, respectively. It can be concluded, that the methodology of data processing is predominant and stronger affects the final results of analysis than the choice of standards for calibration. 4. Conclusions Although optical detection dominates in analytical chemistry for many real applications highly advanced and expensive spectrophotometers are not necessary. For many particular analytical uses simple optical detectors based on extremely cheap optoelectronic components are absolutely sufficient. This statement is especially valid in case of flow analysis, where the systems are developed with the assumption that they are dedicated for particular kind of target analyte, method and sample. In this contribution an extremely cheap photometric PEDD-based detector for Jaffe method operating with low-power source and cheap instrumentation (voltmeter) has been developed and validated. Its utility for real analytics has been confirmed by its implementation into MCFA system and successful application for human urine and serum analysis. It should be pointed out that microsolenoid devices applied in the developed MCFA system also belong to the group of low-budget components useful for construction of flow analysis manifolds. In conclusion, it is worth to indicate further potential biomedical applications of the developed PEDD detector. One of the current trends in clinical urinalysis [26] is the diagnostics of microproteinuria as well as microalbuminuria based on the estimation of creatinine to protein ratio [12,14]. The PEDD concept presented in this paper can be useful for such diagnostics, especially that only recently photometric and fluorimetric [27] as well as turbidimetric and nephelometric [28] PEDD-based detectors dedicated for protein determination have been developed. Another area important from the biomedical point of view are monitors of creatinine removal by artificial kidneys, as this metabolite is established as a model uremic toxin useful for quantitative description of
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hemodialysis efficiency and adequacy [29]. The results presented in this paper evidenced that the determination range offered by the developed PEDD-based detector and MCFA system are also sufficient for such kind of biomedical applications. Acknowledgement These investigations were granted by the Polish National Science Centre (Project Opus NCN no. 2011/01/B/NZ5/00934). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2013.05.052. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29]
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