Proceedings of CUChE Alumni Symposium 2022

On “Circular Economy on Sustainable Basis: The Role of Chemical Engineers”

CUChEAA ISBN: 978-81-954649-1-3

December 2022 P a g e | 48 Volume 2, Issue 1

Design and development of a prototype of POCT device for estimation of protein concentration

in a biological sample

Ravula Rajasekhar

, Thirukumaran Kandasamy

, Siddhartha S. Ghosh

, Tapas Kumar Mandal

1, 4

Centre for the Environment, Indian Institute of Technology Guwahati, Assam, 781039, India

2,3

Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Assam, 781039, India

Department of Chemical Engineering, Indian Institute of Technology Guwahati, Assam, 781039, India

*Email corresponding author: tapasche@iitg.ac.in

Abstract

Quick and precise estimation of total protein concentration is essential in many areas of biology, biochemistry, and

healthcare application to get primary information about various diseases related to the kidney, heart, etc. The Bradford

assay is a simple and accurate method for determining total protein concentrations. It depends on the change in absorbance

limit of Coomassie Brilliant Blue G-250 color from 465 to 595 nm following restriction to denatured total proteins in

solution. We have adopted the Bradford assay route to develop a point of care testing (POCT) device. In this study, a

portable, user-friendly, and highly sensitive optical sensor prototype has been fabricated to measure the unknown total

protein concentration in an aqueous sample. The sensor can measure the total protein concentration from 0-1000 µg/ml,

which enfolds the normal range of total protein concentration present in a healthy human being. A calibration curve has

been developed and compared with UV-Vis spectrophotometer UV-Vis spectroscopic measurement, which proves the

sensor's high accuracy.

Keywords: Total proteins, Detection, Sensor, POCT device

Introduction:

Proteins are the largest and most complex biomolecules,

which play a crucial role in cell structure and functions.

Proteins are made of hundreds of smaller subunits called

amino acids. They are classified into five main

categories based on their function, such as Antibody

(Immunoglobulins), Enzyme (Proteases), Messenger

(Growth hormones), Structural compound (Actin), and

Transporter/storage (Ferritin). Detection of total protein

is crucial for analyzing hundreds of products in the field

of biotechnologies, agriculture, and bioprocess

industries. It is a basis for many research works such as

specific activity determination of enzymes, antibodies,

etc., and also, for diagnostic purposes such as a change

in the total protein levels (depending on the contest)

indicates the abnormal behavior of the body functions or

organs. So, accurate measurement of protein is very

important as their specific activity, and the diagnosis

depends on the accuracy of the total protein

determination.

Most studies are done to detect total protein in a

biological sample. The test for total protein measures the

total amount of two types of proteins found in the human

serum -albumin and globulin [1, 2]. Total proteins are

essential parts of all cells and tissues, and they play the

following vital roles: (a) Albumin helps prevent fluid

from leaking out of blood vessels. (b) Globulins are an

important part of the immune system [2]. The usual

range of total protein concentration in a healthy human

is 600-800 µg/ml [3]. The higher protein concentration is

an abnormal situation. It indicates the following

diseases, such as Chronic inflammation or infection,

including HIV and hepatitis B or C, Multiple myeloma,

and Waldenstrom disease. Similarly, the lower protein

concentration indicates the following diseases:

Proceedings of CUChE Alumni Symposium 2022

On “Circular Economy on Sustainable Basis: The Role of Chemical Engineers”

CUChEAA ISBN: 978-81-954649-1-3

December 2022 P a g e | 49 Volume 2, Issue 1

Agammaglobulinemia, Bleeding (hemorrhage), Burns

(extensive), Glomerulonephritis, Liver disease,

Malabsorption, Malnutrition, Nephrotic syndrome, and

Protein-losing enteropathy. Apart from these two

applications, information on total protein concentration

also gets massive momentum in several fields like

bioprocessing, nutrition, biomedical, and biological

research. As a monitoring system for protein deficiency,

total protein measurement is useful for primary health

care.

The protein sources can be obtained from blood or urine

samples of patients, food products, and bacterial growth

medium (for bioprocess optimization). The predominant

methods used for protein detection are the Lowry

method, Bicinchonic acid (BCA) assay, Bradford assay,

and Ultraviolet spectroscopy [4]. In all of these methods,

protein concentration was measured concerning the color

change during reaction except for the U.V. spectroscopy

method. In the U.V. spectroscopy method, protein

concentration is estimated based on the absorbance of

aromatic amino acids of the protein (tyrosine and

tryptophan) at 275 – 280 nm (UV-Vis spectrophotometer

range) [5]. Since the U.V. spectroscopy method detects

the protein concentration based on the distribution of the

aromatic amino acids in the protein (it varies with

respect to proteins), this method's sensitivity is lesser

than the colorimetric techniques. In the Lawry method,

cupric ions and Folin-Ciocalteau reagent reacts with

proteins and causes the color shift, which can be

measured at 660 nm [6,7]. Likewise, in the Bicinchonic

acid (BCA) assay, cupric is reduced to cuprous ions,

which is measured at 562 nm [8]. In the Bradford assay,

the binding of coomassie dye to the protein causes color

change which absorbs at 595 nm [9,10].

However, all the methods are based on high-end

instruments, which is helpful in the laboratory.

Therefore, it is essential to develop a simple, easy-to-

operate, and portable point of care testing (POCT)

device to measure the total protein concentration in a

biological sample following the Bradford Method. This

study has targeted the development of a LED/LDR

sensor-based portable POCT device functioning on the

principle of an optical sensor.

2. Materials and methods

2.1 Materials

Bradford reagent from Sigma (Cat. No - B6916-500ml),

Bovine serum albumin (BSA) from Himedia (Cat. No -

MB083-25G), Phosphate buffer saline (PBS) from

Himeida (Cat. No – TS1101), UV-Vis

spectrophotometer (Agilent Technologies Cary 60 UV-

Vis), LED Light, light-dependent resistor (LDR),

multimeter, 9 V battery, and U.V. cuvette was purchased

from the local market, IIT Guwahati.

2.2 Design and development of a portable LED/LDR

sensor-based POCT device for the detection of total

proteins

To develop the portable POCT device, an LED light,

LDR, 20×20×20 mm lightproof box, cuvette, 9V battery,

and multimeter are required. Initially, The LED/ LDR

sensor is made by fixing the LED and LDR parallel in

the lightproof box (Fig. 1a), with a gap of 15 mm for

placing the cuvette, as shown in Fig. 1b. Then, the LED

Light is connected to a 9 V battery by a 220 Ω resister to

supply the constant power. The LDR was connected to a

multimeter to measure transmitted light intensity from

the cuvette.

Figure 1: Design and development of a portable

LED/LDR sensor-based POCT device to detect total

protein. (a) A LED/LDR sensor, (b) A portable

arrangement of LED/LDR sensor and analyte chamber

within a light proof box.

3. Experimental procedure

3.1 Preparation of analyte for Bradford assay to detect

the total protein

Bovine serum albumin (BSA) was used as a source to

determine the protein concentration using the present

portable LED/LDR sensor POCT device. The

concentration of BSA (Bovine serum albumin) was

measured with the Bradford reagent (SIGMA, Cat No:

B6919-500ML). Bradford assay helps to determine the

protein concentration based on the absorption spectrum

shift of the Coomassie Brilliant Blue G-250 dye.

Proteins bind with the Coomassie Brilliant Blue G-250

dye in acid conditions and alter its absorption maxima

from 465 nm to 595 nm. The different concentrations of

25, 125, 250, 500, 750, 1000 and 1500 µg/ml BSA

solutions were made from the main stock of 2 mM BSA

by dissolving in 1X PBS (Phosphate Buffer Saline). The

reaction mixture was made by adding 100 µl of BSA

solution and 900 µl of Bradford reagent. The mixture

was incubated for 15 min at room temperature, and the

absorbance was measured using an LED/LDR sensor

and also with a UV-vis spectrophotometer. 1 ml of 1X

PBS was used as a blank. Standard samples were

prepared by using BSA solutions of varying

concentrations (10 mM, 20 mM, 30 mM, 40 mM, 50

mM, and 100 mM).

Proceedings of CUChE Alumni Symposium 2022

On “Circular Economy on Sustainable Basis: The Role of Chemical Engineers”

CUChEAA ISBN: 978-81-954649-1-3

December 2022 P a g e | 50 Volume 2, Issue 1

3.2 Investigation of total protein concentration of the

sample by a portable POCT device

One ml of colored solution mixture (Protein + Bradford

reagent) generated from the Bradford assay was initially

taken into the cuvette by micropipette, as shown in Fig.

2. Then supplied the power to the LED Light. The Light

will pass through the cuvette, and transmitted light

intensity was checked by measuring the LDR resistance.

The multimeter tested the LDR resistance. The process

was repeated separately for all analyte concentrations

such as 25, 125, 250, 500, 750, 1000, and 1500 µg/ml,

and their corresponding resistance values were drawn. A

blank test was also performed to obtain a base resistance

of 5.21 kΩ.

Figure 2: Prototype for detection of total protein in a

biological sample

4. Results and discussion:

The concentration of BSA (Bovine serum albumin) was

measured with the Bradford substance. Bradford assay

helps to determine the protein concentration based on the

absorption spectrum shift of the Coomassie Brilliant

Blue G-250 dye, its absorption maxima from 465 nm to

595 nm. Protein bind with the Coomassie Brilliant Blue

G-250 dye in acidic conditions and will give the Gray-

Blue color, and the color intensity of samples increases

as the protein concentration increase. Based on this

phenomenon, the resistance values of the samples will

vary. The emitted light intensity of the samples

decreases as the sample's color intensity increases. Since

the Light intensity is inversely proposal to the LDR

resistance, the resistance values increase while the

protein concentration increase (As shown in Fig. 3b)

The POCT device performance was investigated over a

broad range of protein concentrations (25-1500 µg/ml.

The sensor POCT device generates a specific resistance

value based on the number of proteins in the samples

and the color intensity. The colored mixture was also

analyzed with a UV-Vis spectrophotometer; in this case,

the absorbance increased as the sample's protein

concentration increased. The calibration curve produces

linear fitting with a regression coefficient (R

) of 0.991.

The calibration plot in Fig. 3a shows a concentration

range versus an absorbance that isn't particularly

quantized. A quantitative estimation is required to

overcome this instrumental-based observation

restriction. An authentic and popular technique, the

LED/LDR sensor-based POCT device technique, has

been adopted here to overcome this scenario. The POCT

device can measure the emitted light intensity in terms

of resistance. In the process, the Light will initially pass

through the solution in the cuvette from the LED source,

and LDR will measure the emitted light intensity. In the

present case, we have extracted all the resistance values

corresponding to the protein concentration of the sample.

The drawn values through the POCT device were

tabulated in Table 1. For a better understanding, the

"resistance vs. concentration of the protein " plot has

also been constructed using the tabulated data, producing

the liner fitting. Fig. 3b gives a perfect linear fitting with

protein concentration range (25-1500) µg/ml with a

corresponding regression coefficient (R

) of 0.994. The

best-fitting equations conveyed below those can be

directly used to predict the concentrations of an

unknown sample -using the contrived POCT device.

Table. 1: Shows the resistance (kΩ) values consistent

with the protein concentration of the sample for the

range of 25-1500 µg/ml.

Resistance (kΩ)

Concentration of proteins (µg/ml)

5.95967

6.27166

125

6.76684

250

7.43206

500

8.69292

750

9.27343

1000

10.9618

1500

Figure 3: Calibration curve for measuring the protein

concentration (a) calibration plot obtained using UV-vis

spectroscopy (b) calibration plot obtained using

LED/LDR sensor-based POCT device (Concentration

(µg/ml) and Resistance(kΩ))

4.1 Assessment of performance of POCT device using

unknown total protein concentration from E. coli Dh5

and validation with UV-vis results

Pretreatment of Bacterial culture (E. coli strain): To

measure the total protein from E. coli Dh5, it has been

treated with the following process. L.B. (Lysogeny

Broth) media are used for bacterial growth. This L.B.

media was made directly by adding 25g of L.B. media

powder into 1000 ml of ddH

O. Then take 40 microliters

Proceedings of CUChE Alumni Symposium 2022

On “Circular Economy on Sustainable Basis: The Role of Chemical Engineers”

CUChEAA ISBN: 978-81-954649-1-3

December 2022 P a g e | 51 Volume 2, Issue 1

of bacterial culture (E. coli strain) and added into 4 ml of

L.B. media. The whole process is done under Laminar

airflow to prevent any contamination. Then allow the

media containing bacterial culture to grow in the

incubator at 37°C for 12 hours (overnight). Centrifuge

the bacterial sample at 10,000 rpm for 2 minutes.

Discard the pellet and take out the supernatant into a

new centrifuge tube with the help of a micropipette. This

sample was used as a mother sample (1). The Bradford

assay uses the mother sample as a sample for measuring

the total protein.

4.2 Evaluation of POCT device using total protein from

E. coli Dh5:

The pretreated E. coli Dh5’s mother sample was taken to

measure the total protein. As described in section 3.1,

the 100 µl of mother sample was taken into a centrifuge

tube, and 900 µl of Bradford reagent was added. It

produced a grey-blue solution that can be used to

measure total protein concentrations with the POCT

device. The color intensity of the mother sample (1) was

analyzed with a POCT device as described in section

3.2, and it is 9.62 kΩ, as displayed in the device in terms

of the resistance.

Furthermore, the mother sample was diluted with PBS

in the following ratios of 1:10 and 1:1. And those

samples (2, 3) were analyzed as described in sections 3.1

and 3.2, and their resulting corresponding resistance

values are 6.4 kΩ and 8.4 kΩ, respectively. All the

samples (1,2,3) were also examined with a U.V.

spectrometer. Based on their results, its corresponding

total protein concentration values are evaluated from the

calibration plots, and resulted are shown in Fig. 4. It is

noted that the POCT device accords to U.V.

spectrometer measurements with an average deviation of

15.78 %. Therefore, it is evidence that the present POCT

portable device is simple and highly efficient for

measuring unknown samples.

5. Concussion

This study has successfully developed a portable POCT

device to estimate the total protein concentration in a

biological sample. This device is advantageous in terms

of low detection time, easy operation, and the use of

trace amounts of reagents compared to the devices

available for lab scale measurement. The response time

of the proposed device is ~1 min. This study was made

for quantitatively estimating unknown protein

concentrations in a biological sample and validated with

U.V. spectrometer results. The results are in good

agreement for unknown samples with an average

deviation of 15.78 %.

Figure 4: Performance of portable POCT device with

respect to UV-Vis spectrophotometry results.

The proposed POCT device can measure the total

protein concentration in a wide range of (25-1500)

µg/ml. For this purpose, a "resistance vs. concentration

of the protein" calibration plot was constructed, which

showed a perfect linear fitting for the whole range of

protein concentration (25-1500 µg/ml) with a high

regression coefficient (R

) of 0.994.

References

[1] Barrett, Kim E., et al. "Ganong’s review of medical

physiology twenty." (2010).

[2] Gonzalez-Quintela, A., et al. "Serum levels of

immunoglobulins (IgG, IgA, IgM) in a general adult

population and their relationship with alcohol

consumption, smoking and common metabolic

abnormalities." Clinical & Experimental Immunology

151.1 (2008): 42-50.

[3] Leeman, Mats, et al. "Proteins and antibodies in

serum, plasma, and whole blood—size characterization

using asymmetrical flow field-flow fractionation (AF4)."

Analytical & bioanalytical chemistry 410.20 (2018):

4867-4873.

[4] Lovrien, Rex, and Daumantas Matulis. "Assays for

total protein." Current protocols in protein science 1.1

(1995): 3-4.

[5] Bencze, W. L., and Karl Schmid. "Determination of

tyrosine and tryptophan in proteins." Analytical

Chemistry 29.8 (1957): 1193-1196.

[6] Lowry, Oliver H. "Protein measurement with the

Folin phenol reagent." J biol Chem 193 (1951): 265-275.

[7] Hartree, E. Fo. "Determination of protein: a

modification of the Lowry method that gives a linear

photometric response." Analytical biochemistry 48.2

(1972): 422-427.

[8] Smith PE, Krohn RI, Hermanson GT, Mallia AK,

Gartner FH, Provenzano M, Fujimoto EK, Goeke NM,

Olson BJ, Klenk DC. "Measurement of protein using

bicinchoninic acid." Analytical biochemistry 150, no. 1

(1985): 76-85.

[9] Bradford, Marion M. "A rapid and sensitive method

for the quantitation of microgram quantities of protein

utilizing the principle of protein-dye binding."

Analytical biochemistry 72.1-2 (1976): 248-254.

Proceedings of CUChE Alumni Symposium 2022

On “Circular Economy on Sustainable Basis: The Role of Chemical Engineers”

CUChEAA ISBN: 978-81-954649-1-3

December 2022 P a g e | 52 Volume 2, Issue 1

[10] Sedmak, J. James, and Sidney E. Grossberg. "A

rapid, sensitive, and versatile assay for protein using

Coomassie brilliant blue G250." Analytical biochemistry

79.1-2 (1977): 544-552.