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Development of test kit for quantitative analysis of urinary albumin with

detection by application on smart mobile phone

..

2558




Development of test kit for quantitative analysis of urinary albumin with
detection by application on smart mobile phone

..

2558

()



2558
220,000

1 1 2557 30 2558
- ..




: (1)
(2)
(3) (4)
2


( 0.74 2.81) 99.6

-

paired t-test (tstat = -1.16, tcri. = 2.78 P = 0.05)

: , , ,

Research Title: Development of test kit for quantitative analysis of urinary albumin with detection
by application on smart mobile phone
Grant:
Faculty of Science, King Mongkut's Institute of Technology Ladkrabang
Fiscal year:
2015 (220,000 THB)
Period:
1 year (1 October 2014 30 September 2015)
Researcher: Asst.Prof.Dr.Nathawut Choengchan
Science
Faculty:
Department: Chemisrtry

ABSTRACT
This work presents method development for urinary albumin analysis by an innovative test kit with
detection by application on smart mobile phone. Detection reaction of albumin is based on its
association with reagent solution. Inside the kit box, there are: (i) cylindrical plastic bottle for urine
sample collection, (ii) plastic syringes for transferring sample and all reagents, (iii) circle-shaped
cassette for solution mixing and (iv) user's manual. Detection procedure was started by transferring
sample and reagent solutions in to the cassette. The cassette was tightened and was shaken. Blue-colored
product was developed. The color was taken a photo through the embedded application in phone/tablet
at 1 min after shaking. The color intensity was evaluated and then converted to the albumin
concentration as mg/L and was displayed on mobile phone. The developed method afforded high
precision (RSD = 0.74 - 2.81 %). Analytical recovery was observed at 99.6 %. The method was applied
to spiked urine from normal volunteers. The results were validated with the results that were obtained by
batchwise method based on the same detection reaction with using UV-Vis. spectrophotometry. There
was no evidence of significant difference by means of paired t-test (tstat = -1.16, tcri. = 2.78 at P = 0.05).
This implies that the kit provided high accuracy.
Keywords : Urinary albumin, Test kit, Mobile phone, Tablet

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III

.. ............................................................................................................. I
.................................................................................................................. II
....................................................................................................................... III
......................................................................................................................................... IV
............................................................................................................................... VII
.................................................................................................................................... VIII
1 .................................................................................................................................. 1
1.1 ........................................................................................ 1
1.2 ............................................................................................. 2
1.3 ............................................................................................ 2
1.4 ......................................................................................................... 3
1.5 .......................................................................................... 3
2 / ........................................ 4
2.1 ......................................................................................................... 4
2.1.1 ............................................................................................................... 4
2.1.2 ................................................................................................................. 4
2.2 (Urine formation) ............................................................................. 5
2.3 .......................................................................................... 6
2.4 ............................................................................................ 7
2.5 .......................................................................................................... 7
2.6 ..................................................................................... 9
2.7 ................................................................................................... 9
2.8 ............................................................................................................ 10
2.8.1 RGB ...................................................................................................... 10
2.8.2 HSV ...................................................................................................... 10
2.8.3 RYB ...................................................................................................... 12
2.9 ............................................................................ 12
2.9.1 .................................................................................................... 12

IV

( )

2.9.2 ................................................................................................. 14
2.10 ............................................................................ 16
3 ................................................................................................................ 18
3.1 ..................................................................................................... 18
3.1.1 ............................................................................................................... 18
3.1.2 ................................................................................. 18
3.2 ................................................................................................... 19
3.2.1 250 mg L-1........................................ 19
3.2.2 TBPE 2.0 x 10-4 mg L-1 ............................................... 19
3.2.3 (0.1mol L-1 Acetic acid 0.1mol L-1 Sodium acetate) .. 19
3.3 .......................................................................................................... 19
3.3.1 .................................................... 19
3.3.2 ........................................................................... 20
3.3.3 ..................................................................... 21
3.4 .................................................................................... 21
3.5 -

................................................................................... 22
4 .......................................................................................................................... 23
4.1 ............................................ 23
4.2 ............................................................. 24
4.3 ............................................................................. 24
4.4 ................................................. 26
4.5
............................................................................ 27
4.6 .............................................................. 29
4.7 ......................................................................... 29
4.8 ..................... 30

( )

5 ........................................................................................ 32
5.1 ................................................................................................ 32
5.2 ................................................................................................................ 33
.................................................................................................................................. 34
........................................................... 36
.............................................................................................. 38

VI

2.1 ...................................................................... 4
2.2 100 () ............................... 6
2.3 .8
4.1 .......................................................................... 29
4.2 %RSD
............................................................................................................ 29
4.3
............................................................................................................ 30
4.4 -
.................................................... 31
4.5 Paired t-test ....... 31

VII

2.1 RGB ........................................................................................................... 10


2.2 HSV ........................................................................................................... 11
2.3 RYB ........................................................................................................... 12
2.4 .............................................................................................................. 13
2.5 .............................................................................................................. 13
2.6 .............................................. 14
2.7 .................................................................................... 14
2.8 ................................................................................... 15
2.9 ........................................................................................ 15
2.10 ............................................... 16
4.1 TBPE
1, 10, 20, 30, 40 50 mg L-1 ..................................................... 23
4.2 1-50 mg L-1 ................ 24
4.3 .............. 25
4.4
.. 21
4.5
....................................................... 23
4.6 pH
60 ......................................................................................................................... 24
4.7
................................................................................. 28
4.8
........................................................................................................................ 28
.1
32 .............................................................................. 36
.2 ( )
32 ........................................................... 37

VIII

( )

.1 .. . ..
....................................................................... 38
.2 2558 .. 38

IX

1.1



( )

150











(image
processing)

(Test kit)

2
: (1) (2)

(3)
(4)
2





1.2
1.2.1

1.2.2

1.3

ion-association
UV-Visible spectrophotometer



(Method validation)
UV-Visible spectrophotometer




3


1.4
1.4.1

1.4.2 ion-association

UV-Visible spectrophotometer
1.4.3

1.4.4
1.4.5

1.4.6 (Method validation)


UV-Visible
spectrophotometer
1.4.7

1.4.8
1.5
1.5.1

15.2
1.5.3

2
/
2.1
2.1.1 [1]

50-60%



30 300
2.1
2.1 [2]

(mg/24hr)
<30
30-299
>300

2.1.2
[3]

-
-
-
- (...)
-
- (Polycystic Kidney Disease)

5
2.1.2.1
1) (Congenital)
(Polycystic kidney disease)
2) (Inflammation)
(Glomerulonephritis)
3) (Infection)
()
4) (Obstruction)

5)
6)
2.2 (Urine formation) [4]

2.2.1.




Colloidal plasma protein

2.2.2




(renal threshold)

6
2.3 [5]
2.2
2.2 100 ()

()
1. Urea Nitrogen
682
2. Urea
1,459
3. Creatinine Nitrogen
36
4. Creatinine
97
5. Uric acid nitrogen
12.30
6. Uric acid
36.90
7. Amino nitrogen
9.70
8. Ammonia nitrogen
57
9. Sodium
212
10. Potassium
137
11. Calcium
19.50
12. Magnesium
11.30
13. Chloride
314
14. Total sulphate
91
15. Inorganic sulphate
83
16. Inorganic phosphate
127

1.
1.1 Amylase (diastase)
1.2 Lactic dyhydrogenate (LDH)
1.3 Leucine amino-peptdase (LAP)
1.4 Urokinase
2.
2.1 Catecholamines
2.2 17-Catosteroids

7
2.3 Hydroxysteroids
2.4 Erytropoietine
2.5 Adenylate cyclase
cyclic AMP
2.6 Prostaglandin

2.7

2.8
2.9 (Melatonin)

2.4 [5]

2.4.1 (Physical examination)

2.4.2 (Chemical examination) -



2.4.3 (Microscopic examination)





2.5 [6]
3
2.5.1 24 (24-hour urine collection)

(Gold standard)
24 (24-hour urinary albumin excretion, UAE)

8
(Urease)

2.5.2 (Spot urine collection)





(Creatinine)
(Albumin
to-creatinine ratio, ACR)
(Creatine)


ACR UAE
42 (Linear
regression)
(Correlation coefficient) 0.952
2.5.3 (Timed-urine collection)
24 12 1
(Albumin excretion rate)
2.3
2.3


24

( )
( ( )
)
< 30
< 30
< 20
30 - 299
30 299
20 199
> 300
> 300
> 200

9
2.6 [6]
D. L. Cohen
[7] 2

-70
30
30
30 0.27
K. Sorensen [8]

2.7



10
2.8 [9]

3
RGB
HLS (hue) (lightness)
(saturation) RGB, HSV (Hue
Saturation Value) HLS (Hue Lightness Saturation)
2.8.1 RGB
RGB
Additive CRT (Cathode ray tube)
RGB RGBCIE RGBNTSC
2.8.1.1 RGB CIE
CIE (Commission International de lEclairage)
700 nm 546.1 nm 435.8 nm
2.9.1.2 RGB NTSC
NTSC (National Television System Committee)
CRT CRT

2.1 RGB
2.8.2 HSV
HSV (Hue Saturation Value) Hue, Saturation
Value Hue ( ) 0 255
Hue 0 Hue

11
256 = 0
120 240 Hue RGB
(2.1)
(2.2)
(2.3)

2.2 HSV
Hue 0
0 hue ( ) 0
Hue -
0
Saturation Saturation 0 Hue
Saturation 255 Saturation

(2.4)

Value

(2.5)

12
2.8.3 RYB
RYB [SSubtractive CColor: The RYB
R Primaries (Opaque PPigments)
3 (red) (blue) (yellow
w) (Suubtractive


Colorr)




(Subttractive Coloor Mixing)

22.3 RYB
22.9 [10]
(Digitaal Image Proocessing)



2.9.1
(Digital
(
Imagge)

(Bitmap IImage)

(Raaster Graphiics)
(Vector Image)

(PPixels)




2.99.1.1 (Biitmap Image)

((Pixels)








13



2.4 [11]
2.9.1.2 (Vector Image)






2.5 [11]

14

2.6 [11]
2.9.2 (Image Type) [12]
4

2.9.2.1 (Intensity Images or Gray Scale Image)


(Pixel)
(Gray Scale Gray Level )
2.6 8
225

2.7 [12]
2.9.2.2 (Color Image)
3
2.7

15

2.8 [12]
2.9.2.3 (Binary Image)
(Binary) 1 1 0 1
0 (Text)
(Finger Print) 2.8

2.9 [12]
2.9.2.4
(Index Number) (Color Table)

3 2.9

16

2.10
2.10


G. L. Luque [13]
carbon-nanotube electrode T. K. Chritopoulos [14]
ion-selective electrode
ligand ligand-ion
selective electrode
N. T. Deftereos [15]
potassium permanganate polyphosphoric acid
Flow Injection Analysis
(FIA) [16] 2006 X. Chen
[17] fluorescamin

Sequential Injection Analysis
(SIA) [18] FIA
Y. F. Li [19]
o-phthalaldehyde sulfanilic acid


colorimetry K. Watla-iad [20]
tetrabromophenolphthalein (TBPE)
607 nm 2006 W. Qin [21]

17
o-nitrophenylfluorone
Mo(V)
Z. X. Guo [22]
533 nm
Dibromohydroxyphynylfluorone Mo(V)
(Image processing)

[23, 24] 2011 L. Lahuerta Zamora
[23]
Image J
Flame absorption spectrometer
2013 Sarun Sumriddetchkajorn [24]
software

18

3

3.1
3.1.1
1. (Human serum albumin) Aldrich, USA

2. (Tetrabromophenolphthaline
ethyl ester, TBPE) Aldrich, USA
3. X-100 (Triton X-100) Aldrich, USA
4. (Ethanol) Mallinckrodt
5. (Acetic acid) Mallinckrodt
6. (Sodium Acetate) Rankem
7. (Deionized-distill water)
3.1.2
1.
2.
3.
4.
5.
6.
7.
8.
9.
10. Vortex- Genie Z, USA
11. pH-Metrohm 827 pH Lab meter, USA
12. - Jasco V630 ,USA
13. Sumsung galaxy S3
14. Sumsung galaxy Note 10.1
15.
15.1. 1

19
15.2. 1
15.3. 2
15.4. 5
15.5.
15.6.
15.7.
3.2
3.2.1 250 mg L-1
0.0125
50.00
3.2.2 TBPE 2.0 x 10-4mol L-1
TBPE 0.0070 50
Triton X-100 0.1

3.2.3 (0.1 mol L-1 Acetic acid 0.1 mol L-1 Sodium acetate)
- 0.1 mol L-1 Sodium acetate 0.4101
50.00
- 0.1 mol L-1 Acetic acid Stock acetic acid 99.5 %
1.43 250.00
0.1 mol L-1 Acetic acid 245.58 0.1 mol L-1
Sodium acetate 45.00 pH 3.0
3.3
3.3.1
3.3.1.1 1, 10, 20, 30 40 mg L-1
250 mg L-1 0.20, 2.00, 4.00, 6.00, 8.00
10
5, 50, 100, 150, 200 mg L-1

20
3.3.1.2 5 mg L-1 0.50
TBPE 2.0 x 10-4mol L-1 0.50 40
1.50 50 Cuvette
2
3.3.1.3 12 50,
100, 150, 200, 250 mg L-1 2
3.3.2
3.3.2.1 (TBPE 2 x 10-4 M, Buffer pH 3.0)

1) TBPE 2 x10-4 mol L-1 5 mL


20.00 pH 3.0 15 mL 30
2) 2.00 mL
0.50 30 2
600
3)
1, 7, 14, 21 30
4)
30 mg L-1
3.3.2.2
1) TBPE 2x10-4 mol L-1 20.00
pH 3.0 20.00 2

2) TBPE 2 x10-4 mol L-1 pH 3.0

3) TBPE 2 x10-4 mol L-1 0.50 mL
30 mg L-1 0.50 mL 30
pH 3.0 1.50 30
2

21
4)
1, 7, 14, 21, 28, 30, 45, 60 90
5) TBPE 2 x10-4 mol L-1
pH 3.0 TBPE 2 x10-4mol L-1
pH 3.0
3.3.3
3.3.3.1
1) TBPE 2 x10-4 mol L-1 20.00
pH 3.0 20.00 2

2) TBPE 2 x10-4 mol L-1 20.00
pH 3.0 20.00 2
4
3) TBPE 2 x10-4 mol L-1 0.50
30 mg L-1 0.50 30
pH 3.0 1.50 30
2
4)
1, 7, 14, 21, 28, 30, 45, 60 90
5) TBPE 2 x10-4 mol L-1
pH 3.0 TBPE 2 x10-4 mol L-1
pH 3.0 4
3.3.3.2 pH
1) pH 3.3.3.1

2) pH
1, 7, 14, 21, 28, 30, 45, 60 90

22
3.4
3.4.1
3.4.2 Matlab
3.4.3
3.4.4
3.4.5
3.4.6
3.5 -

3.5.1
3.5.1.1 0.25
25 100
3.5.2 -
3.5.2.1 100 0.50
TBPE 2.0 x 10-4mol L-1 0.50 40
1.50 50 Cuvette
2
3.5.2.2
3.5.3

3.5.3.1 100 0.50


TBPE 2.0 x 10-4mol L-1 0.50
40 1.50 50
2

23

44.1



TBBPE Tritonn X-100 pH 3.0 (4.1)
((4.2) W. Kannchana [20]
TBPPE + Triton X-100 + Bufffer pH 3.0

(TTBPE-H)m
(4.1)
( )

x(TBPE-HH)m + Humann Serum Albuumin (HSA)X+X (HSAX+. (TBPE-) X)m + xH+ (4.2)
( )
( )

((TBPE) triton
t X-100
0.1 3

1--50 mg L-1
UV-VVisible spectrrophotometerr 4.1


4.1 TBPE

11, 10, 20, 30, 40 50 mg


m L-1

24
4.1 TBPE
600 nm

44.2

TBPE triton x-1000 pH 3.0


1-50 mg
m L-1
4.22

4.2

1-50 mg L-1
4.2 1--50 mg L-1
(Coeefficient of DDeterminationn) 0.99903

44.3
4.3.1

(TBBPE 2 x 10-4 M,
M Buffer pHH 3.0)
TBPE 2 x 10-4 M pH 3.0


4.3

25

( )
4.3

( )

30 mg L-1

TBPE
4.3

600

15

+ ( )
TBPE
T

4.3.2

TBPE
(Fresshly prepareed)

4.4
4

26

4.4

( )
( )


4..4

600


44.4

4.4.1


44.5


4.5
( ) ( )

27
4

6000

4.5
4

4
4.4.2
pH



pH 4.6
4

4.6 pH
60
4.6
pHH 4



44.5


4.5.1

4.7

28

4.7

4.5.2

HSV model






4.8

4.8

29
4.6
(Recovery) 100

4.1
4.1

Dilution factor
-1

100

(mg L )

(mg L-1)

24.49
24.49

32.02
22.96

% Recovery
130.7
93.8


100 % recovery
100
4.7
4.7.1

(1, 10, 20, 30, 40 50 mg L-1) 5
4.2
4.2 %RSD

(mg L-1)

(mg L-1)

SD

%RSD

1
10
20
30
40
50

1.03
9.51
18.63
27.96
39.12
50.35

0.022
0.267
0.564
0.206
0.748
0.945

2.10
2.81
3.03
0.74
1.91
1.88

30
4.2
5%
4.7.2
30 mg L-1

30 mg L-1
30 mg L-1
4.3
4.3

1
2
Mean
%Recovery

(mg L-1)

Spiked sample
33.562
32.993
33.2775

Sample
0.216
0.214
0.215
99.61

Standard
34.022
32.364
33.193

4.3
99.61 %


4.8

4.8.1 -

-

4.4
4.5

31
4.4 -

(mg L-1)

1
2
3
4
5

UV-Vis
Mean
22.86
28.10
27.57
30.57
30.24

Application
SD
0.66
2.79
0.62
1.25
2.00

Mean
24.31
33.74
30.15
29.16
29.39

SD
2.80
0.37
3.86
1.33
0.40

4.5 Paired t-test


t-Test: Paired Two Sample for Means
-1.166
t Stat
2.776
t Critical two-tail
4.4 4.5 2
95 %

32

5

5.1

1-50 mg L-1
Abs. = 0.0071[Albumin] + 0.1533 (r2) 0.9903
TBPE

TBPE


4

4 pH
4


4


mg L-1


100
93.8 %RSD
0.74 3.03
99.61 -


33

5.2
1.
2.

34


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32

32

(.) (.) 4 2557
( )

.1
32

37

.2 ( )
32

38

.1 2558
.. ..
" Hilight" " "
. 24-25 ..58

.1 .. . ..

.2 2558