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