Journal of Clinical Laboratory Analysis 28: 104–109 (2014)

Simplified Microchip Electrophoresis for Rapid Separation of Urine Proteins Hongwei Song,1 Huimin Wang,1† Saoqing Ju,1 Qinghui Jin,2 Chunping Jia,2 and Hui Cong1 ∗ 1

Medical Laboratory Center, Affiliated Hospital of Nantong University, Jiangsu Province, P.R China Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Science, Shanghai, P.R. China

2

Background: Urine protein test has been ring was 2.68% and 2.24%, and RSD of widely used in clinics, but to determine the the peak area was 5.85% and 4.96%, retype of proteinuria is usually difficult due to spectively. The linear detection range was technical limitations. Methods: In the cur- 1.0–15.0 g/l for purified human albumin and rent study, a rapid and simple method to 1.0–10.0 g/l for human transferrin, with the separate and determine urine proteins by a same detection limit (S/N = 3) of 0.4 g/l. microchip electrophoresis (ME) system has Finally, comparing to conventional agarose been developed in which only 4 min are re- gel electrophoresis, the same results were quired. Results: Optimal separation condi- obtained by using ME by testing clinical tions have been established by using 15 s samples including 60 selective proteinuria, injection time at 500 and 1,500 V separa- 105 nonselective proteinuria, and 6 overtion voltage in 75 mmol/l borate buffer con- flow proteinuria. Conclusion: This newly estaining 0.8 mmol/l calcium lactate and 1% tablished ME could have broad applicaϕ ethylamine (pH 10.55). Relative standard tions to determine the type of proteinuria in deviation (RSD) of migration time with pu- clinics. J. Clin. Lab. Anal. 28:104–109,  C 2014 Wiley Periodicals, Inc. rified human albumin and human transfer- 2014. Key words: microfluidic chip electrophoresis; urine proteins; UV detection; calcium lactate; ethylamine; selectivity of proteinuria

INTRODUCTION A urine protein test is to detect the presence of protein in a person’s urine (1, 2). In healthy individuals, there is only very low concentration of protein in urine (3, 4). But in renal patients, the concentration of urine protein will increase considerably (5–7). Several simple urine protein tests including sulfosalicylic acid and colorimetric test strips (“dipstick” tests) and commonly used in clinics to help diagnose renal and related diseases. However, these 31 methods can only detect the presence of protein in urine, but cannot provide the information of which type of proteinuria it is. Agarose gel electrophoresis (AGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis are the most common routine methods to separate small amounts of protein in biological fluids (8). Both methods require fixation, staining, washing, and densitometric scanning after separation. These procedures are technically demanding and laborious. Recently, with the rapid development of  C 2014 Wiley Periodicals, Inc.

microchip analytical technology, microchip electrophoresis (ME) has attracted much attention due to its substantial advantages over conventional analytical technologies such as high speed, low sample loading and buffer consumption, easy integration, and miniaturization (9–12). ME has been demonstrated to be a powerful tool for efficient separation of DNA fragments (13, 14), amino acids (15, 16), and drugs (17). As far as protein analysis is concerned, ME separation of proteins is currently limited Grant sponsor: State 863 High Technology R & D Project of China; Grant numbers: 2002AA404310 and 2004AA404252; Grant sponsor: Technology Project of Jiangsu; Grant number: H200216. † These authors contributed equally to this work. ∗ Correspondence to: Hui Cong, Medical Laboratory Center, Affiliated Hospital of Nantong University, Jiangsu Province, P.R. China, 226001. E-mail: [email protected]

Received 21 July 2012; Accepted 3 June 2013 DOI 10.1002/jcla.21651 Published online in Wiley Online Library (wileyonlinelibrary.com).

Simplified ME for Rapid Separation of Urine Proteins

Fig. 1. Channel pattern of the chip. Buffer (B), sample (S), sample waste (SW), detection point (DP), buffer waste (BW).

to purified proteins (18–21). There is no report concerning using microfluidic chip to separate urine proteins. In this work, we present a rapid and simple method to separate urine proteins with ME. By testing different experimental conditions, the optimal parameters were established and the duration of the analytical run takes less than 4 min. Finally, we have compared ME and conventional AGE and the same results were obtained by analyzing 248 urine samples.

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length was 42 mm. The diameter of all the reservoirs was 2.5 mm. Purified human albumin, human transferrin, antihuman immunoglobulin G (IgG), and calcium lactate were purchased from Sigma (St. Louis, MO). Water was prepared with Milli-Q (Millipore, Bedford, MA) purified water instruments. All buffers were routinely degassed in an ultrasonic bath and filtered through 0.22 μm membrane filters (Millipore). Buffer preparations: (a) 100 mM phosphate buffer (pH 8.0): 5.3 ml solution A (200 mM, 2.760 g NaH2 PO4 dissolved in 100 ml H2 O) mixed with 94.7 ml solution B (200 mM, 3.561 g Na2 HPO4 dissolved in 100 ml H2 O); (b) 100 mM borate buffer (pH 10.0): detailed preparation method will be described in detail in the following text; (c) 25 mM borax-NaOH buffer (pH 10.1): 0.954 g borax and 0.8 g NaOH dissolved in 100 ml H2 O; (d) 100 mM sodium barbital-hydrochloric acid (HCl) buffer (pH 9.6): 2.062 g sodium barbital and 0.7 ml HCl (0.1 M) contained in 100 ml H2 O; (e) 100 mM TrisHCl buffer (pH 9.1): 1.121 g Tris and 8 ml HCl (0.1 M) contained in 100 mL H2 O; (f) 100 mM 2-amino-2-methyl1,β-propanediol (AM2 P)-HCl buffer (pH 10.0): 50 ml solution A (0.2 mol/l AM2 P, 0.105 g AM2 P dissolved in 100 ml H2 O) mixed with 2.3 ml HCl, then added water to a total volume of 100 ml. All the buffers pH adjusted with NaOH (all buffer regents were purchased from Shanghai Chemical Regent Corporation).

EXPERIMENTAL Instruments and Reagents

Patient Samples and Normal Controls

The chip electrophoresis apparatus was a product of Shanghai Institute of Microsystem and Information Technology (Shanghai, China) (22), consisting of an optical module, a control module, and a detection module. The 214 nm UV light was obtained by a hologram grating and focused the separation channel to detect the presence of protein. The light absorbed by a sample was gathered by a photomultiplier tube (Hamamatsu, Beijing, China) and converted to electric signals, which were then processed by a filtering and amplifying circuit and input to a PC by an acquisition card (Hengshengyuan, Beijing, China). A Visual Basic program was used to orderly control the electrophoresis process, display the electrophoretic curves, and store the data automatically. Comparative results (standard electrophoretic separation) were obtained, 61 with REP Automated Electrophoresis Instrument (Helena Company, Texas, USA). The chips used were produced by Shanghai Institute of Microsystem and Information Technology (Shanghai, China) and made of quartz from Shanghai DuPont Photomasks, Inc (23). The chip layout was shown in Figure 1. The microchannel dimensions were 30 μm deep and 100 μm wide at half-depth. The distance between the center of the sample reservoir and intersection was 4 mm and the effective separating

A total of 248 clinical urine samples were collected and divided into the patient group (178 samples) and the control group (70 samples). In patient group, the samples were positive for urine proteins (more than 2+ with qualitative by 10A urine regent strip method and most of which came from patients with renal disease, and in the control group all were with negative for urine proteins. Electrophoresis and Determination Prior to injecting the sample, the microchannels of the chip were flushed with 1 M NaOH for 1 min first, then rinsed with DI water for 1 min, and flushed with the separation buffer for 1 min finally. After all the microchannels of the chip were conditioned, we dropped the sample solution into sample reservoir, the other reservoirs filled with the running buffer, and applied a voltage of +500 V on sample reservoir and sample waste reservoir for 40 s to fill the intersection. Then +2,000 V voltage was applied on buffer reservoir and buffer waste reservoir to inject the plug into the separation channel. When a voltage of +500 V applied on the sample and sample waster reservoirs, the buffer and buffer waster reservoirs were floated. And when a voltage of +2,000V J. Clin. Lab. Anal.

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Fig. 2. Electropherograms of a urine sample with NS by chip electrophoresis with not adding ethylamine (A) and adding 1% ϕ ethylamine (B) in the running buffer. The electrophoretic conditions: 75 mmol/l borate buffer containing 0.8 mmol/l calcium lactate with pH 10.55, injection voltage of 500 V for 30 s, separation voltage of 2,000 V for 4 min.

applied on the buffer and buffer waster reservoirs, the sample and sample waster reservoirs were floated. Agarose Gel Electrophoresis AGE was performed on a REP automated gel electrophoresis apparatus (Helena) using the REP SP300 plate kit (Helena). Briefly, 3 μl urine sample was applied to each well (30 wells/gel), and allowed to diffuse for 1 min before separation at 350 V at 21◦ C for 8 min in a tris-barbital/MOPS buffer with calcium lactate and a stabilizer. Gels were dried at 60◦ C for 11 min, stained with 0.5% (w/v) acid blue stain, and destained with 0.3% (w/v) citric acid for 5 min before air-drying at 20◦ C. The electrophoretic profile was obtained by densitometric scanning of the gel at 525 nm on an EDC scanning denistometer. The total analysis time was about 45 min. RESULTS AND DISCUSSION Selection of Buffers Different buffers would influence the resolution (Rs) and detection limits of the samples in ME. We have compared different buffers in the ME system. According to migration time stability, peak shapes, and the detection sensitivity, we chose the borate buffer in the subsequent experiments. As buffer concentrations would directly affect the resolution and speed of analytical materials, the effects of concentrations in the range of 25–150 mmol/l borate buffer on the efficiency of chip electrophoresis separation were compared by using the same mixed model protein solutions as the above. In the range of 25–50 mmol/l of the running buffer, two model proteins could not be separated completely, but when the concentration of running buffer was in the range of 100–150 mmol/l, the electroosmotic flow (EOF) decreased and migration time delayed, the separation current and the output Joule heat increased J. Clin. Lab. Anal.

Fig. 3. Diagrams of reproducibility with model proteins by quartz chip electrophoresis. The concentrations of purified human albumin and human transferrin were both 5.0 g/l. The electrophoretic conditions: 75 mmol/l borate buffer containing 0.8 mmol/l calcium lactate and 1% ϕ ethylamine (pH 10.55), injection voltage of 500 V for 15 s, separation voltage of 1,500 V for 4 min.

owing to the increased ionic strength. So we chose 75 mmol/l borate buffer in the subsequent research. As the walls of the quartz chip channels bore negative charges, resulting in absorption of proteins on the walls during separation (20), to avoid protein adsorption, we used extreme pH. Having known that too little EOF in extremely acidic pH was disadvantageous to sample injection and separation, we chose extremely alkali pH. We prepared a series of 75 mmol/l borate buffer with 0.2 mmol/l boric acid and 1 mol/l NaOH and their pH values

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TABLE 1. The Regression Equations and Detection Limitsa Compound Human albumin Human transferrin

Regression equationb

Correlation coefficient

Linear range (g/l)

Detection limit (g/l)

y = 0.06498 + 0.15565x y = –0.04386 + 0.12985x

0.9996 0.9999

1.0∼15.0 1.0∼10.0

0.4 0.4

a The b In

electrophoretic conditions are the same as in Figure 3. the regression equation, the x value is the concentration of analytes (g/l), the y value is the peak area.

changed the property of the charge on the surface of the microchannel wall, EOF decreased, and the migration time and resolution increased. Purified human albumin and human transferrin underwent baseline separation at 0.8 mmol/l concentration of calcium lactate, so this concentration was chosen in the following experiments. Effects of Ethylamine on Separation

Fig. 4. Electropherogram of mixed solution containing three model proteins whose concentrations were all 4.0 g/l. For the electrophoretic conditions, see Figure 3.

were 9.98, 10.34, 10.41, 10.55, 10.67, 10.84, 11.22, 11.51, 12.05, and 12.95, respectively. The effects of buffer pH on the efficiency of ME separation were tested on the mixed model protein solution of purified human albumin and human transferrin. In the range of pH 9.98–10.55, with the pH value raised, the resolution increased, but in the range of pH 11.22–12.05, the results went to the opposite. There was no significant change in migration time in the range of pH 9.98–11.51, but when the pH value was higher than 12.05, the migration time delayed. We chose pH 10.55 in the following experiments. Effects of Calcium Lactate on Separation Addition of calcium lactate to the running buffer with CE could enhance the resolution of albumin and globulin (24). We prepared a series of calcium lactate solutions with concentrations at 0.1, 0.5, 0.8, 1.0, and 2.0 mmol/l, respectively. The effects of calcium lactate concentrations on the efficiency of chip electrophoresis separation were tested on the mixed model protein solutions of purified human albumin and human transferrin. The results showed that with the increased calcium lactate concentration, the migration time and resolution of the mixed model proteins increased. As Ca2+ underwent ion exchange with H+ on the silicon hydroxy of chip microchannel wall surface and

ME often needs to reduce protein adsorption or control the EOF rate by using a dynamic or static wall coating (25). Verzola et al. (26) reported that the adsorption between proteins and capillary wall decreased by 90% in the presence of 10 mmol/l o,o-dimethyl phosphoramidothionate (DMPAT). Gysler et al. (6) used CZE to monitor chemotherapy-induced proteinuria and found that the number of protein bands increased by addition of 2 mmol/l putrescine to 100 mmol/l borate buffer with pH 9.0 in order to decrease the EOF. Considering that the principle of chip electrophoresis was similar to CE, we adopted ethylamine as an additive and studied the effects of its different concentrations from 0.5% to 2% ϕ on the efficiency of separating urine proteins with chip electrophoresis. With the concentration of 0.5% ϕ ethylamine in the running buffer, four bands were separated from the urine sample with nephropathy syndrome (NS). When its concentration went up to 1% ϕ, four bands achieved baseline separation, whereas its concentration reached 2% ϕ, migration time was further delayed and peak breadth became widened. So 1% ϕ ethylamine was chosen as an additive. The electropherograms of a urine sample with NS by ME without ethylamine (A) or with 1% ϕ ethylamine (B) in the running buffer were shown in Figure 2. Selection of Separation Voltage and Injection Time Separation voltage would obviously affect resolution and migration time and was correlative with the EOF. As ME mostly used the EOF to drive protein bands (21), the EOF would increase and sample migration time would shorten with the increased separation voltage. We studied the effects of separation voltage in the range of 1,200– 2,000 V on the efficiency, resolution, and migration time. When separation voltage declined from 2,000 to 1,500 V, J. Clin. Lab. Anal.

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Fig. 5. Electropherograms of three kinds of proteinurias with microfluidic chip and gray scanning results of the photos of slab gel. For the electrophoretic conditions, see Figure 5. (A, B) Selective proteinuria; (C, D) nonselective proteinuria; (E, F) overflow proteinuria; (A, C, E) with microfluidic chip electrophoresis; (B, D, F) with agarose gel electrophoresis.

resolution improved, but when separation voltage was declined from 1,500 to 1,200 V, migration time was delayed, resolution was not enhanced, and the protein peak breadth was widened. So 1,500 V was chosen as the separation voltage in the experiments. Electric injection was actualized by adopting crossinjector initially, later “T,” double “T,” and narrow injections. Because crossing structure was the most classical and most commonly used in chip electrophoresis, we chose crossing injection in the experiment. The effects of injection time in the range of 10–30 s were tested on mixed model proteins solution of purified human albumin and human transferrin with injection voltage 500 V and separation voltage 1,500 V adopted in electric injection. When injection time was reduced from 30 to 20 s, electropherograms basically did not change. But when injection time was reduced to 15 s, a baseline separation was achieved for the two model proteins. While injection time was reduced to 10 s, migration time was unstable with poor reproducibility. So we chose 15 s as injection time.

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Repeatability, Linearity, and Detection Limits Under the optimum conditions mentioned above, we then detected the repeatability of ME separation by repeating the experiments for five times. Relative standard deviation (RSD) of migration time with purified human albumin and human transferrin was 2.68% and 2.24%, and RSD of peak area, respectively, was 5.85% and 4.96%, respectively. The diagrams of repeatability of chip electrophoresis were shown in Figure 3. Linear ranges of purified human albumin and human transferrin were tested and their regression equations were calculated by peak area and their concentration. From Table 1, we can see that the linear ranges for human albumin and human transferrin were 1.0–15.0 g/l and 1.0–10.0 g/l, respectively, and the detection limits were 0.4 g/l for both proteins. We then tested a ME protein separation by using mixed purified human albumin, human transferrin, and

Simplified ME for Rapid Separation of Urine Proteins

human IgG. As Figure 4 showed, a baseline separation was achieved for the three model proteins. Compared with other substances, creatinine and vitamin C have relatively high concentration and are special representative substances in human urine, so we want to study the interference of them to this method. We added certain amount of creatinine or vitamin C into the urine from patients with urinary proteins, and the results show that when the concentrations of creatinine were up to 250 mg/l or vitamin C up to 500 mg/l had no interference to this method. Study Clinical Urine Samples In 1960, Blainly et al. put forward the concept of the selectivity of proteinuria and made it as an index for judging severity extent of glomerular lesions by measuring the ratio of middle and high molecular weight proteins in urine by electrophoresis. In our study, we judged the selectivity of proteinuria by peak area ratio of albumin and globulin. If the value was more than 5, it was selective proteinuria, whereas it was nonselective. A total of 248 preprocessed clinical urine samples were tested with ME, and the results of this assay were compared with American Helena AGE. The control group containing 70 urine samples in which no protein peak was detected. The patient group contained 178 urine samples, of which no protein peak was detected for 7 samples, 60 were selective proteinuria with primary protein of albumin, 105 were nonselective proteinuria with various molecular weight proteins, and 6 were overflow proteinuria. (Overflow proteinuria is a result of the filtering process of the kidneys being overwhelmed by an overproduction of protein, most commonly from the disease multiple myeloma.) American Helena AGE obtained the same results as the ME. The seven false-positive proteinuria samples were from the patients with no renal or urinary system diseases. Urine protein was tested by pH error principle, and falsepositive results were affected by icterus, alkaline urine, medicine. Representative electropherograms of the each type of proteinuria with ME and gray scanning results of the photos of slab gel were shown in Figure 5. We could see that the order of protein peaks for ME and AGE was reversed and this is because the ME mainly drove protein bands by the EOF. CONCLUSIONS In the current study, we have described a novel ME method to determine the type of proteinuria. The results showed were full consistent with standard American He-

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lena AGE. This assay was rapid, simple, and convenient, with electrophoretic time only 4 min and good reproducibility. The electrophoresis apparatus and detection system were cheap and microfluidic chip could be repeated. In conclusion, this newly established ME provides a useful clinical tool with broad potential applications for the diagnosis, treatment, and prognosis of renal and related diseases. ACKNOWLEDGMENTS The authors thank Shanghai Institute of Microsystem and Information Technology for providing devices and microchip fabrications, and Shanghai Changzheng Hospital and Shanghai Renji Hospital for supplying proteinuria samples. REFERENCES 1. Barratt J, Topham P. CMAJ 2007;177:361–368. 2. Ottiger C, Savoca R, Yurtsever H, Huber AR. Clin Chem Lab Med 2006;44:1347–1354. 3. Larson TS. Mayo Clin Proc 1994;69:1154–1158. 4. Maruhn D, Fuchs I, Mues G, Bock KD. Clin Chem 1976;22:1567– 1574. 5. Beetham R, Cattell WR. Ann Clin Biochem 1993;30:425–434. 6. Gysler J, Schunack W, Jaehde U. J Chromatogr B 1999;721:207– 216. 7. Jones BR, Bhalla RB, Mladek J, et al. Clin Pharmacol Ther 1980;27:557–562. 8. Nauck M, Winkler K, Wittmann C, Mayer H, Luley C, M¨arz W, Wieland H. Clin Chem 1995;41:731–738. 9. Petersen JR, Okorodudu AO, Mohammad A, Payne DA. Clin Chim Acta 2003;330:1–30. 10. Jabeen R, Payne D, Wiktorowicz J, Mohammad A, Petersen J. Electrophoresis 2006;27:2413–2438. 11. Bossuvt X. Clin Chem Lab Med 2003;41:762–772. 12. Bruin GJ. Electrophoresis 2000;21:3931–3951. 13. Castoldi M, Schmidt S, Benes V, et al. RNA 2006;245:913–920. 14. Phillips TM, Wellner E. J Chromatogr A 2006;1111:106–111. 15. Xu B, Feng XJ, Xu YZ, Du W, Luo QM, Feng LB. Anal Bioanal Chem 2009;394:1911–1917. 16. Wang AJ, Feng JJ, Fan J. J Chromatogr A 2008;1192:173–179. 17. Guihen E, Sisk GD, Scully NM, Glennon JD. Electrophoresis 2006;27:2338–2347. 18. Root BE, Zhang B, Barron AE. Electrophoresis 2009;30:2117– 2122. 19. Kavran BG, Stefan S. J Nanosci Nanotechnol 2009;9:2645–2650. 20. Doherty ES, Meagher RJ, Albarghouthi MN, Barron AE. Electrophoresis 2003;24:34–54. 21. Badal MY, Wong M, Chiem N, Moosavi HS, Harrison DJ. J Chromatogr A 2002;947:277–286. 22. Wang H, Wang HM, Jin QH, Cong H, Zhuang GS, Zhao JL, Sun CL, Song HW, Wang W. Electrophoresis 2008;29:1932–1941. 23. Zhuang GS, Liu J, Jia CP, Jin QH, Zhao JL, Wang HM. J Sep Sci 2007;30:1350–1356. 24. Jenkins MA, Kulinskaya E, Martin HD, Guerin MD. J Chromatogr B 1995;672:241–251. 25. Dolnik V. Electrophoresis 1999;20:3106–3115. 26. Verzola B, Gelfi C, Righetti PG. J Chromatogr A 2000;868:85–99.

J. Clin. Lab. Anal.

Simplified microchip electrophoresis for rapid separation of urine proteins.

Urine protein test has been widely used in clinics, but to determine the type of proteinuria is usually difficult due to technical limitations...
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