J. Flow Injection Anal., Vol. 28, No. 2 (2011) 120–123

Flow Injection Determination of Urinary Protein Using Fluorescence
Quenching of Tetraiodofluorescein
Tadao Sakai1,*, Norio Teshima1, Toshiyuki Kato1, Shuji Katoh2, Weena Siangproh3
1

Department of Applied Chemistry, Aichi Institute of Technology, 1247 Yachigusa, Yakusa-cho,
Toyota 470-0392, Japan2
Murakami Memorial Hospital, Asahi University, Hashimoto-cho, Gifu 500-8523, Japan3
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Sukhumvit 23, Wattana,
Bangkok 10110, Thailand
Abstract
A flow injection analysis system with spectrofluorimetric detection has been developed for trace protein in urine. The protein assay
is based on the ion-association of protein and tetraiodofluorescein (TIF) in the presence of Triton X-100 at pH 3.2. Although the
TIF-protein ion associate produces red color compounds, fluorescence quenching caused by bonding with protein was measured at
560 nm (excitation: 313 nm). The quenching is applied to the protein determination. Linearity range was up to 10 mg/L bovine serum
albumin and the correlation coefficient was 0.999. The detection limit (S/N = 3) was 0.6 mg/L with RSD 0.134% (n = 4). The FI
system is available for rapid screening in diabetic diagnosis.

Keywords Urinary protein, tetraiodofluorescein, fluorescence quenching, flow injection
1. Introduction
Proteins synthesized in the liver are the building blocks for all
body parts. Usually, most proteins are too big to pass through thekidneys’ filters into the urine unless the kidneys are damaged.
Protein is excreted as a renal proteinuria in nephropathy anddiabetic nephropathy. As a result, measurement of proteinsexcreted in urine is an important and necessary to the diabetes
diagnosis. In the clinical laboratory, the first line test paper
impregnating the dyestuff is commonly used for the rapid
decision of renal disease. The visible detecting technique using
the first line test paper is simple and convenient in handling,
however, the detection range of protein is semi-quantitative andthe measurable concentrations are classified as follows; (–) < 15
mg/dL, (±) 15 – 30 mg/dL, (+) > 30 mg/dL, (++) > 100 mg/dL,(+++) > 300 mg/dL. The color development on the test paper
relates to amount of abnormal products in the urine. However, it
does not offer accurate analytical value with two or moresignificant figures. Yoshimoto et al. have proposed the visual,
simple and sensitive analytical method for the protein detection[1,2] and Sakai et al. have reported a simple and visible method
for urinary protein [3].
On the other hand, simple and accurate spectrophotometricdeterminations of urinary albumin using bromophenol blue [4,5]have been reported. And, Fujita et al. have reported highly
sensitive spectrophotometric methods for urinary protein withdye-metal complexes in the micelle media [6–9]. Bromocresol
green and bromocresol purple were used for the serum albumin
determination [10] and bromothymol blue has been proposed forthe determination of serum albumin [11]. Recently, spot test of
urinary protein using erythrosine B and a membrane film was
proposed for very sensitive detection method [12]. However,these methods mentioned above have been performed by thebatchwise technique and the procedure needs much time andlarger reagents consumption.
Flow injection analysis (FIA) developed by Ruzicka andHansen is a versatile and practical technique [13]. The
technology has been widely used in environmental, clinical and*Corresponding author. E-mail address: tadsakai@aitech.ac.jp

pharmaceutical analyses because the method permits automatic,
rapid and sensitive analysis compared with the batchwise
method. FIA with Coomassie brilliant blue G-250 was used todetermine rapidly protein in urine [14] and also, high-sensitivity
flow method with micro-flow plunger pumps [15] wasdemonstrated for the serum albumin determination. However,the sample throughput was only 15/h.
In a previous paper, we reported two channel FIA system with
TBPE·H (tetrabromophenolphthalein ethyl ester)-protein
associate (λmax 610 nm) formed at pH 3.8. The calibration graph
was linear in the range of 1.5 – 12 mg/dL albumin and thesample throughput was 30/h [16]. A flow injection system is asemi-automation method when compared with a completely
automatic sequential injection (SI) proposed in 1990 [17].
Moreover, the consumption of reagents and sample in SIA isusually smaller than that in FIA.
An accurate and automated SI method has been proposed forstepwise determination of protein and glucose in urine [18]because it was necessary to determine protein and glucose forthe diagnosis in the clinical laboratory. Human serum albumin(HSA) was determined using an ion association reaction of HSA
with TBPE·H, and glucose was indirectly determined bymeasuring hydrogen peroxide generated by the oxidation of
glucose in the presence of glucose oxidase using iron-catalyzed
oxidation reaction of p-anisidine by H2O2. As a result, the linear
ranges were up to 10 mg/dL HSA and 12.5 mg/dL glucose. The
sample throughput was 6/h.
The American Diabetes Association (ADA) recommends that“the measurement of urinary albumin excretion is suitable for a
24-h urine collection” [19]. However, this method is extremely
time-consuming.
To
avoid
this
tediousness,thealbumin/creatinine ratio (ACR) for a random urine specimen was
proposed as a more convenient detection of micro-albuminuria[20]. We proposed an automated SIA system for successive
determinations of albumin and creatinine and their ratio in
urinary samples based on eosin Y binding and Jaffe’s reaction,
respectively [21]. The calibration graph was linear up to 20 mg/L
for albumin. The relative standard deviation (n = 10) was 2.49 %for 20 mg/L albumin. The sample throughput was 20/h. However,these procedures are not rapid and sensitive.

– 120 –

In this study, we found that tetraiodofluorescein (TIF) reacts
with albumin to show quenching and the negative intensity is in
proportion to the albumin concentration. The proposed FIA
system using TIF-BSA binding method is more rapid andsensitive compared with previous results by FIA and SIA
methods and the method is applicable to real samples.

2.3. Preparation of urine samples

2. Experimental

3. Results and discussion

2.1. Reagents

3.1. Fluorescent spectra

All chemicals and reagents were analytical reagent grade andwere used without further purification. The deionized water usedthroughout the experiments was purified by an AdvanceGSH-210 apparatus. A standard bovine serum albumin (BSA)(1000 mg/L) was prepared by dissolving 0.0250 g of BSA (Wako
Pure Chemical Co., Japan) in 25 mL of water. The BSA standard
solutions should be prepared freshly every time. Working
standard solutions were prepared by accurate dilution of thestock solution with 0.03 M dihydrogenphosphate. TIF (5  10–3
M) was prepared by dissolving 0.22 g of TIF (Tokyo Kasei
Kogyo, Japan) in 50 mL of water. The working concentrations of
the dye were prepared by suitable dilution. A Triton X-100
solution (0.5%) was prepared by dissolving 0.5 g oft-octylphenoxypolyethanol (Sigma Chemicals Japan) in 100 mLof water. Buffer solution (pH 3.2) was prepared by mixing 0.4 M
sodium acetate and 0.4 M acetic acid.

Fluorescent spectra were measured for 5 mg/L BSA standard
solution with 0.4 M buffer (pH 3.2), 2.0  10–5 M TIF and
0.048% Triton X-100 by the batchwise procedure. We found theexcitation wavelength was at 313 nm and emission wavelength
was at 560 nm. The fluorescent intensity with formation of theion associate, BSA-TIF, decreased in proposition to theconcentration of BSA.

2.2. Appratus
JASCO, Model FP-6200 spectrofluorimeter (Japan) was used
for measuring fluorescence intensity.. The manifold of the flow
injection system is shown in Fig. 1. The dual micro-pump (FIAInstruments, Model 201) was used to deliver carrier (CS) andreagent solution (RS). The flow lines were of Teflon tubing (0.5mm inner diameter). CS is 0.4 M acetate buffer and RS is the
mixture of 2.0  10–5 M TIF and 0.08% Triton X-100. An aliquot(200 μL) sample solution is injected into the carrier stream by a
six-way injection volume. The flow rate is 1.0 mL/min.
Excitation wavelength is 313 nm and emission wavelength is
560 nm for measuring fluorescence intensity.

S
CS

V
RC
P

Ｄ
D

W

RS
Ｄ
Rec

Fig. 1 Schematic diagram of the flow system for thedetermination of albumin.
CS: 0.2 M acetate buffer (pH3.2)
RS: A mixed solution of 1.010–5 M TIF and 0.04% TX-100
P: 1.0 mL min–1 (0.5 mL min–1 each line)
S: BSA (0 – 10 ppm) or sample
V: Six-way injection valve
RC: Reaction coil (0.3 m, 0.5 mm i.d.)
D: Fluorescence detector (λex = 313 nm; λem = 560 nm)
Rec: Recorder
W: Waste

The urine samples supplied from diabetic patients were kept at–20C. Before analysis, the urine samples were filtered toremove small particles. Then, the filtered solution was diluted at
400-fold with 0.4 M acetate buffer solution.

3.2. FIA study for determination of albumin
3.2.1. Effect of reaction coil
For 5 mg/L BSA, 0.025 M buffer, 1  10–5 M TIF and 0.04%
Triton X-100 solutions were used and the length of the reaction
coil as shown in Fig.1 was varied in the range of 0.3 m – 2.0 m.
The shortest 0.3 m length gave the strong intensity because ofquick formation of the BSA-TIF associate. With increasing thelength, the intensity decreased gradually. A 0.3 m-tubing wasused in the system.
3.2.2. Effect of pH
Under the same condition described above, the effect of pH
for the ion associate formation was investigated in the range of
2.8 – 4.2. Above pH 3.1, the negative intensity increased,
however, the intensity decreased over 3.4. In the system, pH 3.2
was chosen.
3.2.3. Effect of flow rate
The flow rate was varied from 1.0 mL/min to 3.5 mL/min.
Over 2.5 mL/min, the peak height was decreased slightly
because of dispersion. In this study, 1 mL/min was used.
3.2.4. Effects of TIF and TritonX-100 concentrations
For 5 mg/L BSA, TIF concentration was varied in the range of6  10–6 M – 2.0  10–5 M. The negative intensity decreased up
to 1.0  10–5 M. Over its concentration, the baseline was noisy.
And also, when quenching of TIF was used, the higher
concentration was not suitable because of larger blank signal. 1.0
 10–5 M TIF was chosen in this study. On the other hand, theeffect of Triton X-100 was studied in the range of 0.02% –0.05%. At 0.02%, the intensity was small, however, the intensity
increased with increasing Triton X-100 concentration. Over
0.03%, the optimum and constant peak height was obtained asshown in Fig. 2. In this study, 0.04% Triton X-100 was chosen.
3.3. Analytical characteristics
Calibration graph for BSA was established under the optimal
conditions. Fig. 3 shows typical flow signals for BSA. The
linearity was obtained in the range of 0 – 10 mg/L with r2 0.999(y = 4.22x – 0.178, y = fluorescent intensity, x = albumin
concentration in mg/L). The detection of limit (S/N = 3) was 0.6
mg/L. When TBPEH was used for the dye-binding method, thelinearity was obtained in the range of 1.5 – 120 mg/L in FIA– 121 –

510min
min

50
45

CBSA

40

10 mg L–1

1

ΔRFI
ΔＲＦＩ

５ RFI

30

10 RFI35

325

5 mg L–1

20

5

15
10
5
0
0.02

0.025

0.03

0.035

0.04

0.045

10 mg L–1

0.05

CTritonＴｘ－１００/％
X-100 / %Fig. 2 Effect of Triton X-100 concentration.

Fig. 3 Typical flow signals for BSA standards.

[16] with RSD 1.2% and/or in the range of 0 – 100 mg/L with
SIA [18]. On the other hand, the calibration graph was obtainedin the range of 0 – 20 mg/L with another SIA [21]. In this study,
RSD for 10 mg/L albumin (n = 4) was 0.134%. The obtained
RSD was small compared with other SIA, 2.7% in Ref. 18 and2.5% in Ref. 21. Although the sample throughputs were 6/h in
Ref. 18, 18/h in Ref. 21 and 30/h in Ref. 16, 60/h sample
throughput in the proposed FI system was obtained.
Consequently, the proposed method is rapid, sensitive andreproducible compared with other flow-based techniques.
3.4. Interference study
For the determination of albumin, various amounts of foreigncompounds were added in the 5 mg/L BSA standard solution andtheir interference was examined. The results are shown in Table
1. The tolerance limit was defined as the interference thatyielded a relative error less than 5% when compared the
response obtained from the standard concentration. In the normal
urine, the chloride and urea contents are worthy of remark andthe average concentrations in the urine are 4751 for chloride ion
and 18200 mg/L for urea, respectively [18]. Sodium chloride,
urea and creatinine did not interfere in the protein determination
up to 100 mg/L, 500 mg/L and 10 mg/L. Other foreign species(potassium, ammonium, magnesium, calcium ions, glucose)normally present at low concentrations in the real urine samples
did not interfere.

Table 1 Tolerance limits of foreign compounds for thedetermination of 5 mg L–1 BSA
CompoundTolerance limit /mg L–1NaCl

100 Na+ 39 mg L–1; Cl– 61 mg L–1

KCl
MgCl2

50 K+ 26.2 mg L–1; Cl– 23.8 mg L–1
500 Mg2+ 127 mg L–1; Cl– 373 mg L–1

CaCl2

100 Ca2+ 36 mg L–1; Cl– 64 mg L–1

NH4Cl
Urea
GlucoseCreatinine

100 NH4+ 33.6 mg L–1; Cl– 66.4 mg L–1
500
300
10

The average concentration levels in an ordinary person’s urine:Na+ 2940 mg L–1; Cl– 4780 mg L–1; K+ 2346 mg L–1; Urea 18200
mg L–1; Uric acid 420 mg L–1; Creatinine 1960 mg L–1.

Compound

The influence of dilution effects was investigated to ensure
accurate quantitation. Urine samples may be diluted with buffer
solution to obtain the concentration within the examined range.
The recovery tests were carried out to evaluate the effects for thedetermination of 5 mg/L BSA in a real urinary sample. The
results are shown in Table 2. As a result, 200-fold dilution
eliminated interferences. When distilled water was used for the
dilution of sample, we found a small ghost peak. Consequently,
we used 400-fold dilution with 0.025 M buffer solution for thereal sample before analysis.
3.6. Application to real samples
The proposed method was applied to evaluate albumin in
urinary samples from diabetic individuals. And also, theTolerance limit /R–1

Table 2 The recoveries of the determination of 5 mg L–1 BSA ina real urinary sample using different dilution factors

mg L

Dilution factor
20
50
100
200
300
400
500
600

NaCl
KCl

3.5. Dilution effectRemarks

MgCl2

CaCl2
CompoundRecovery / %
180
133
115
104
98.4
97.1
97.1
100

– 122 –

50 K

500 M

100
Tolerance limit/ CR

correlation between the proposed method and Bradford–1 method[22] was investigated (Table 3). A paired t-test with 5 degrees of
freedom was performed on the data obtained. The experimental
t-value between two pairs of the methods was 1.357 for the 95%
confidence interval (2.571) and it was significantly small.
Accordingly, the measurements are reliable and acceptable.

NH4Cl

100 N

mg L

100 N

Urea
NaCl

500
100 N

Glucose
KCl

300
50 K

Table 3 Determination of albumin in urine taken from diabetic
patients

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95
110
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110
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Acknowledgements[20] American Diabetes Assoication: Clinical practicerecommendations 2001: Diabetic nephropathy, Diabetes
The authors gratefully acknowledge the financial support ofCare 24 (2001) S69-S72.
this study by Grants-in-Aid for Scientific Research Nos. [21] W. Siangproh, N. Teshima, T. Sakai, S. Kotoh, O.
21550093 (to T.S.) and 22550085 (to N.T.) from Japan Society
Chailapakul, Talanta, 79, 1111 (2009).
of the Promotion of Science.
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