J. Biomedical Science and Engineering, 2010, 3, 285-290
doi:10.4236/jbise.2010.33038 Published Online March 2010 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online March 2010 in SciRes. http://www.scirp.org/journal/jbise
Z-Scan technique: To measure the total protein and albumin
in blood
A.N.Dhinaa, P.K.Palanisamy*
Department of Physics, Anna University Chennai, Chennai, India.
Received 19 December 2009; revised 28 December 2009; accepted 4 January 2010.
ABSTRACT
Z-scan technique is an effective tool for measuring
the optical nonlinearity of the materials. By using this
technique the measurement was made for total pro-
tein and albumin. The nonlinear refractive index of
the total protein and albumin were found to vary
linearly with concentration. Hence by calculating the
nonlinear refractive index it is possible to measure
their concentration in the sample. The values meas-
ured thus are found in equivalence with conventional
colorimetric method.
Keywords: Z-scan Technique; Nonlinear Refractive
Index; Total Protein; Albumin
1. INTRODUCTION
Protein is an essential nutrient made up of building-
block chemicals called amino acids. Protein provides
energy and is needed for the body to make new cells, to
maintain and rebuild muscles, to carry other nutrients, to
act as messengers in the body, and to support the
immune system. A total serum protein test measures the
total amount of protein in the blood. It also measures the
amounts of two major groups of proteins in the blood:
albumin and globulin.
Albumin is made mainly in the liver. It helps keep to
the blood from leaking out of blood vessels. Albumin
also helps to carry some medicines and other substances
through the blood and is important for tissue growth and
healing.
Globulin is made up of different proteins called alpha,
beta, and gamma types. Some globulins are made by the
liver, while others are made by the immune system.
Certain globulins bind with hemoglobin. Other globulins
transport metals such as iron in the blood and help fight
infection.
Low total protein levels can suggest a liver disorder, a
kidney disorder, or a disorder in which protein is not
digested or absorbed properly. Low levels may be seen
in severe malnutrition and with conditions that cause
malabsorption, such as Celiac disease or inflammatory
bowel disease (IBD). High total protein levels may be
seen with chronic inflammation or infections such as
viral hepatitis or HIV. They may be caused by bone
marrow disorders such as multiple myeloma.
Measurements of protein may reflect liver disease,
nutritional state, kidney disease and others. A decreased
value of total protein may indicate liver or kidney
disease. If levels of albumin are low, there is a possi-
bility of primary liver disease, kidney disease, tissue
damage or inflammation, and malnutrition [1,2]. In
chronic liver diseases like “cirrhosis” or “nephrotic synd-
rome” the level is decreased. Poor nutrition or protein
catabolism may cause “hypoalbuminaemia”. Measurement
of serum-total protein is useful in conditions relating to
changes in plasma or fluid volumes, such as shock and
dehydration. In these conditions concentration of
serum-total protein is elevated indicating hemoconcen-
tration. Haemodilution is reflected as relative hypoprotei-
nemia, which occurs with water intoxication or salt
retention syndrome, during massive intravenous infusions.
The most widely accepted assays so far for proteins
are the Biuret [3], Lowry [4], Bradford [5,6], Bromop-
henol Blue [7] and Bromocresol Green[8] methods. In
this Biuret reaction is highly susceptible to interference
by non-protein substances [9,10,11,12]. The bromocresol
green method for determination of serum albumin is the
most specific and sensitive of the dye binding techniques
[13]. The glyoxylic acid method measures tryptophan
content which represents 8-10% albumin and 90-91%
globulin. Since the bromocresol green method is specific
and simple, it is the method of choice for albumin
determination [14].
The Z-scan technique was extended to study the opti-
cal nonlinearity has been reported for LDL-Choles-
terol [15,16]. Some more reports are on characterization
of lipids in body fluid [17,18], study of the nonlinear
refraction of vitreous humor in human and rabbit [19],
determination of nonlinear refractive index of retinal
derivatives [20]. In this present investigation total protein
and albumin are subjected to the Z-scan technique to cal-

286 A. N. Dhinaa et al. / J. Biomedical Science and Engineering 3 (2010) 285-290
culate the nonlinear refractive index (n2). Already work
has been done on measurement of glucose [21], total
cholesterol and triglycerides [22].
The single beam Z-scan analysis, which was developed
by Mansoor Sheik Bahae et al. [23], is a simple and
effective tool for determining nonlinear optical proper-
ties of materials [24,25,26,27]. This approach has been
now a day widely used in optical characterization of differ-
ent materials. Nonlinear refractive index is proportional to
the real part of the third-order susceptibility Re[χ(3)]. Ba-
sically, the Z-scan method consists in translating a non-
linear sample through the focal plane of a tightly focused
Gaussian laser beam and monitoring the changes in the
far field intensity pattern. For a purely refractive nonlin-
earity, the light field induces an intensity dependent non-
linear phase and, as consequence of the transverse Gaus-
sian intensity profile, the sample presents a lens-like
behavior. The induced self-phase modulation has the ten-
dency of defocusing or recollimating the incident beam,
depending on its Z position with respect to the focal
plane. By monitoring the transmittance change through a
small circular aperture placed at the far field position, it
is possible to determine the nonlinear refractive index. In
the present study, we have measured total protein and al-
bumin levels in blood by calculating the nonlinear re-
fractive index (n2) value using a single beam Z-scan
method.
2. METHODOLOGY
2.1. Preparation of Total Protein Sample
For sample preparation (Total Protein-Biuret method - a
kit supplied by Transasia Bio-medicals Ltd, Baddi,
Himachal Pradesh, India) was used. To 20 microliter of
the serum one milliliter of total protein reagent was
added and incubated for 10 minutes at 37 oC. The principles
involved for this reaction is that the peptide bonds of
protein react with copper II ions in alkaline solution to
form blue-violet complex (Biuret reaction). Each copper
ion complexes with 5 or 6 peptide bonds. Tartrate is
added as a stabilizer whilst Iodide is used to prevent
auto-reduction of the alkaline copper complex. The color
formed is proportional to the protein concentration.
2.2. Preparation of Albumin Sample
For sample preparation (Albumin-BCG method - a kit
supplied by Transasia Bio-medicals Ltd, Baddi, Himachal
Pradesh, India) was used. To 10 microliter of the serum
one milliliter of albumin reagent was added and incubated
for 1 minute at 37oC. The principle involved in this reac-
tion is that the albumin binds with Bromocresol green
(BCG) at pH 4.2 causing a shift in absorbance of the
yellow BCG dye. The Blue green color formed is propor-
tional to the concentration of albumin.
2.3. Nonlinear Refractive Index
The Z-scan experiments were performed using a 532 nm
Nd: YAG (SHG) CW laser beam (COHERENT–Compass
215M-50 diode-pumped laser) and He-Ne laser beam
(RESEARCH ELECTRO OPTICS–30995 cylindrical
helium-neon laser) focused by a lens of 35 mm focal length.
The experimental set up is shown in Figure 1.
A typical closed-aperture Z-scan curve for the stan-
dard total protein solution at incident intensity Iο =
7.824 kW/cm2. Likewise the Z-scan curve for standard
albumin solution at incident intensity Iο = 1.758 kW/cm2.
This normalized transmittance curves are characterized
by a pre-focal peak followed by a post-focal valley. This
implies that the nonlinear refractive indices of total pro-
tein, albumins are negative (n2 < 0). The defocusing ef-
fect shown in Z-scan curve can be attributed to a thermal
nonlinearity resulting from absorption of radiation at 532
nm and 633 nm respectively. Localized absorption of a
tightly focused beam propagating through an absorbing
sample medium produces a spatial distribution of tem-
perature in the sample solution and consequently, a spa-
tial variation of the refractive index, that acts as a thermal
lens resulting in phase distortion of the propagating beam.
The nonlinear refractive index (n2) is calculated using
the standard relations [18].
0.25
p - v0
ΔT = 0.406 (1 S) ΔФ (1)
Where ΔTp -v can be defined as the difference be-
tween the normalized peak and valley transmittances
pv
(TT )

, 0 ΔФ is the on-axis phase shift at the focus.
The linear transmittance of the aperture is given by
22
1(2
aa
S - exp - r / w ) (2)
where ra is the radius of the aperture and wa is the beam
radius at the aperture.
0
2
0eff
nkI L

 (3)
where n2 is the nonlinear refractive index, k is the wave
number( 2
λ
k

) and
L
aser
B
ea
m
Solution
Aperture
Detector
-Z +Z
Figure 1. Experimental setup for Z-scan technique.

A. N. Dhinaa et al. / J. Biomedical Science and Engineering 3 (2010) 285-290
287
3. RESULTS AND DISCUSSION
L
1e
α
eff
L
 


3.1. Measurement of Absorbance Spectra
02
0
2P
Iw

 is defined as the peak intensity within the
sample at the focus. L is the thickness of the sample, α is
the linear absorption coefficient.
The absorption spectra were measured using UV-Vis
spectrophotometer (SHIMADZU- UV-2401PC), and the
spectra for both total protein and albumin were found to
be broad banded as depicted in Figure 2. Hence for fur-
ther study 532 nm Nd:YAG laser for total protein and
633 nm He-Ne laser for albumin were used.
An additional experiment was performed with a con-
ventional colorimetric method following the standard
procedure of A. G. Gornall et al. [3] and R. L. Rodkly
et al. [8] for total protein and albumin samples respec-
tively. This involves measurement of optical density
variation with respect to concentration. These results are
compared with the results calculated with the Z-scan
technique.
3.2. Measurement of Nonlinear Refractive Indices
The results of typical Z-scan normalized transmittance
measurement for total protein and albumin are shown in
Figure 3. As the concentration of the total protein and
albumin increases, the normalized transmittance peak
increases whereas the valley decreases respectively. The
graph in Figure 4 (a) and (b) shows that the ΔTp-v value
linearly increases with concentration of standard total
protein and albumin solutions. Similarly in Figure 4 (c)
and (d) refractive index value linearly increases with con-
centration of standard total protein and albumin solutions.
2.4. Statistical Analysis
The error involved in the measurements was determined
by t test, P < 0.01.These statistical analysis was con-
ducted using SPSS commercial statistical package (SPSS,
version 10.0 for windows, SPSS Inc., Chicago, U.S.A). In addition experiment based on optical density is
400 450 500 550 600 650 700
0.1
0.2
0.3
0.4
0.5 (a)
Absorbance (arb.units)
Wavelength (nm)
400 450 500 550 600 650 700
0.0
0.5
1.0
1.5
2.0
2.5 (b)
Absorbance (arb.units)
Wavelength (nm)
Figure 2. UV-Vis Spectra of standard (a) total protein (b) albumin with reagent.
-10 -50510
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Normalised Transmittance
Z (mm)
TP (4g/dl)
TP (6g/dl)
TP (8g/dl)
-10 -50510
0.7
0.8
0.9
1.0
1.1
1.2
1.3
Normalised Transmittance
Z (mm)
Albumin (2g/dl)
Albumin (4g/dl)
Albumin (6g/dl)
Figure 3. Z-scan data of the standard total protein (TP) and albumin.
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288 A. N. Dhinaa et al. / J. Biomedical Science and Engineering 3 (2010) 285-290
4681012
0.2
0.4
0.6
0.8
1.0
1.2 (a)
R2=0.99707
Y=0.12453x-0.2796
 Tp-v
Concentration of total protein (g)
123456
0.1
0.2
0.3
0.4
0.5
0.6 (b)
R2=0.98172
Y=0.08939x+0.03901
 Tp-v
Concentration of albumin (g)
4 6 81012
10
20
30
40 (c)
Y=3.85085x-8.86715
R2=0.99539
n2x10-8cm2/W
Concentration of total protein (g)
123456
0
2
4
6
8
10
(d)
R2=0.98648
Y=1.50678x+0.47998
n2x10-8cm2/W
Concentration of albumin (g)
Figure 4. Linear variation of T p-v and nonlinear refractive index (n2) with concentration of total protein (a,c)
and albumin (b,d) by Z-scan method.
4681012
0.2
0.3
0.4
0.5
0.6
0.7 (a)
R2=0.99954
Y=0.0545x+0.04
Optical Density (arb.units)
Concentration of total protein (g)
123456
0.1
0.2
0.3
0.4
0.5
0.6 (b)
R2=0.99763
Y=0.09343x+0.05133
Optical Density (arb.units)
Concentration of albumin (g)
Figure 5. Linear variation of optical density of total protein (a) and albumin (b) by colorimetric method.
given in Figure 5 (a) and (b). The experiments were
repeated five times and the mean value of the nonlinear
refractive index (n2) was calculated from the normalized
transmittance values. This calculated value was assumed
to be the standard for measurement of unknown total
protein and albumin content present in blood sample.
This can be arrived by plotting a linear graph of total
protein and albumin concentration Vs nonlinear refrac-
tive index. The nonlinear refractive index value was first
measured against the reagent blank solution. The calibra-
tion was made with the conventional colori-metric method
and the results are tabulated in Table 1 for total protein
and in Table 3 for albumin. The normal level of total pro-
tein in serum is in the range of 6–8.3 g/dl, and serum al-
bumin normal level is in the range of 3.2–5 g/dl.
For estimating the total protein and albumin levels,
one need not plot full Z-scan curve every time. Once,
experimental setup explained above is established, one
needs to note down peak and valley values of the trans-
mittance curve translating the sample holder continu-

A. N. Dhinaa et al. / J. Biomedical Science and Engineering 3 (2010) 285-290
289
JBiSE
ously along Z-axis. The difference in these two values
Tp–Tv, ׀ΔФ0׀ when substituted in Equation (3) yields
the nonlinear refractive index value.
Consequently by the results of Z-scan method, we in-
fer that the n2 value is to be in the range of 13.90 ± 1.98
to 23.01 × 10-8 cm2/W for normal level of total protein in
serum. Likewise, n2 value for normal level of albumin in
serum is to be in the range of 5.26 to 8.16 ± 0.98 × 10-8
cm2/W.
3.3. Valuation with Conventional Method
Many trials were performed to measure the total protein
and albumin level with Z-scan method. The blood sam-
ples were collected from five volunteers. We could see
that the results arrived are in good agreement with those
of the conventional colorimetric method for total protein
as shown in Table 2 and for albumin Table 4. Hence we
could clearly ascertain that the Z-scan method is on par
with the conventional colorimetric method. By calculat-
ing the total protein and albumin values we can also
calculate the globulin level in serum. (Globulin = Total
Protein–Albumin) is tabulated in Table 5.
Table 1. Nonlinear refractive index (n2) values for standard
total protein.
Standard total protein
Concentration (g/dl)
Nonlinear refractive index
n2 10-8 (cm2/W)
4 06.32 ± 0.74
6 13.90 ± 1.98
8 23.91 ± 1.79
10 30.81 ± 1.53
12 34.97 ± 1.89
Table 2. Comparative analysis of serum total protein meas-
urement using colorimetric method and Z-scan method.
Concentration of total protein (g/dl)
Total
Protein level Colorimetric method Z-scan method
Normal 6.33 6.22
Normal 6.83 6.90
Normal 6.50 6.54
Normal 7.83 7.79
Normal 7.33 7.26
Table 3. Nonlinear refractive index (n2) values for standard albumin.
Standard albumin
concentration (g/dl)
Nonlinear refractive index
n2 10-8 (cm2/W)
1 1.60 ± 0.58
2 3.81 ± 0.73
3 5.10 ± 0.85
4 7.42 ± 0.81
5 8.16 ± 0.98
6 9.03 ± 0.65
Table 4. Comparative analysis of serum albumin measurement
using colorimetric method and Z-scan method.
Concentration of albumin (g/dl)
Albumin
level Colorimetric method Z-scan method
Normal 3.42 3.49
Normal 3.85 3.78
Normal 3.68 3.75
Normal 4.20 4.13
Normal 4.02 4.08
Table 5. Globulin concentration calculated from colorimetric
method and Z-scan method.
Concentration of globulin (g/dl)
Colorimetric method Z-scan method
2.91 2.73
2.98 3.12
2.82 2.79
3.63 3.66
3.31 3.18
4. CONCLUSIONS
The Z-scan measurements indicate that the total pro-
tein’s and albumin’s standard sample and serum sample
exhibit nonlinear optical properties. We have measured
the nonlinear refractive index values for total protein and
albumin present in the serum sample by Z-scan method
with 532 nm Nd:YAG CW laser and 633 nm He-Ne laser
respectively. Comparative analysis of these values with
the one obtained by conventional colorimetric method
shows that they are in good agreement. Hence, apart
from existing techniques, Z-scan technique can also be
used for the measurement bioanalytes in serum.
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