DEVELOPMENT AND CHARACTERIZATION OF ANALYTICAL

METHODS FOR ESTIMATION OF SOME BIOMOLECULES .

Paper II

Review of Literature

Prepared by -
Pooja Deshpande
Research Scholar
UIOP, Ptrsu , Raipur (C.G)

1
Biomolecule or biological molecule is the term used for molecule or ions that are present in
organisms essential to some biological process such as cell division , morphogenesis or
development .

Biomolecules include large macromolecules such as Proteins , Carbohydrates, Lipids and Nucleic
acid.

For example, Insulin is an important polypeptide hormone that regulates carbohydrate metabolism. 1

2
 Types of Biomolecules

• PROTEIN -Made from building blocks

called amino acids.

• CARBOHYDRATES- Stores of chemical

energy building materials for biological
construction.
• LIPIDS- Group of nonpolar biological
molecules , ability to dissolve in organic
solvents.
• NUCLEIC ACID - Small biomolecules
essential to all known forms of life.

• Hormones - Mediator molecules released in

one part of the body but regulates the activity of
cells in other parts of the body . 2, 31

3
 PROTEINS
• Macromolecules that carry out virtually all of cell’s activities.
• The Building Blocks of Proteins– AMINO ACIDS sequence .
• 20 different amino acids are commonly used in the construction of proteins,
whether from a virus or a human.
• All amino acids have a carboxyl group and an amino group. 2

4
 Carbohydrates

• Carbohydrates (or glycans )

• Simple sugars (or monosaccharides) and all larger molecules.
• Sugar building blocks.
• Function stores of chemical energy and as durable building materials for
biological construction
• Each sugar molecule consists of a backbone of carbon atoms linked
together in a linear array by single bonds. Each of the carbon atoms of the
backbone is linked to a single hydroxyl group, except for one that bears a
carbonyl (C= O) group 2

5
 Lipids

• Lipids are a diverse group of nonpolar biological molecules whose

common properties are their ability to dissolve in organic solvents, such as
chloroform or benzene
• Lipids of importance in cellular function include
 fats
 steroids
 phospholipids. 2

6
Table : Types of Biomolecules 2, 31
PROTEINS NUCLEIC ACID
CARBOHYDRATE LIPIDS HORMONES
S
•Alanine •Monosaccharides • Fats •Human DNA
•Asparagine •Disaccharides • Steroids growth RNA
•Aspartic •Trisaccharides • Phospholipids hormone
acid •Tetrasaccharides •Thyroid
•Glutamic •Polysaccharides Hormone
acid •FSH
•Histidine •LH
•Isoleucine •Prolactin
•Leucine •ACTH
•Lysine •Melanocyte
•Methionine stimulating
•Phenylalani Hormone
ne
•Threonine
•Tryptophan
•Valine

7
 Estimation of Biomolecules
• PROTEINS
1. Biuret method-The reagent used in this method are Copper sulfate,
Sodium hydroxide, and Potassium Sodium Tartrate. Protein reacts with this
alkaline copper complex and color changes to violet. The protein can then
be estimated by reading the absorption at 540nm.
2. BCA assay - This method is highly sensitive and detects proteins at a low
concentration of 1 µg. In this method, Copper ions bind to Nitrogens in
protein and the complex is then bound to bicinchoninic acid resulting in
the change of color to purple depending on protein concentration
3. Lowry assay - It detects proteins at low concentrations of 2-5 µg. In this
method, first, the copper ions are reduced under alkaline conditions and
forms a complex with peptide bonds of the protein. This complex then
reduces Folin-Ciocalteau reagent and results in the change in color to deep
blue and absorption can be measured at 650-750nm. 3

8
 CARBOHYDRATES

1. Molisch’s test –
ᾳ Napthol + conc . H2SO4 Purple colour

2. Fehling test –
Solution of Carbohydrate + equal quantity of Fehling sol A & B heating Brick red ppt 4

9
 Lipids

• Iodine value – wt of iodine absorbed by 100 parts by weight of sample of

fat or oil .
• Saponification value – no of mg of KOH required to neutralize the fatty
acids resulting from complete hydrolysis of 1 gm of sample .
• Hydroxyl value – No of mg of KOH req to nutralize acetic acid for
acetylation with 1ml of sample or fat
• Acetyl value – No. of mg of KOH required to neutralize acetic acid when 1
gm of sample acetylated oil is saponified. 5

10
• Acid value – No of mg of KOH required to neutalize the free acids present
in 1 gm of sample of fat or oil .
• Peroxide Value – Measure of peroxides present in sample – less than 10
mEq/kg in fresh samples of oil
• Rancidity test- Malonaldehyde are increased due to rancidity – detected by
phloroglucinol produces red colour with oxidised fat. 5

11
 ANALYTICAL METHODS FOR ESTIMATION OF
BIOMOLECULES
PROTEIN CARBOHYDRAT LIPIDS NUCLEIC
ES ACID
•Kjeldahl method •TLC •UV-visible •Isolation of
•ELISA •GC •Infrared 24 DNA/RNA
•Western blotting •HPLC15 •Nuclear Magnetic •Electrophoresis22
•PCR12 •Gravimetry Resonance •RNA – cDNA
•MALDI – TOF •Colorimetry •X-ray absorption15 conversion
20,28
•Polarimetry •TLC •PCR12
•HPTLC •Infrared •GC •qPCR13
•HPLC •Immunoassay •HPLC 14 •Sanger method
•UV •Next generation
•FTIR 17 sequencing (NGS)
•Size exclusion •Restriction
chromatography 9
fragment length
•MS 7 polymorphism
(RFLP) 6

12
 Review in Related Field

1. FOR PROTEIN
• Paula Hong et al( 2012) described that the use and number of biotherapeutics has increased significantly. For these
largely protein-based therapies, the quantitation of aggregates is of particular concern given their potential effect on
efficacy and immunogenicity. This need has renewed interest in size-exclusion chromatography (SEC). 9
• Igor A. Kaltashov et al (2012) studied Biological mass spectrometry (MS) a variety of approaches to study structure
and behavior of complex protein drugs and has already become a default tool for characterizing the covalent structure of
protein therapeutics, including sequence and post-translational modifications. 10
• Cristina et al (2013) focused primarily on the cereal-based commodities available for developing gluten free blends,
considering naturally gluten-free cereals in addition to oats, and recent transgenic approaches for developing cereals
free of immunotoxic gluten. The methods of analysis includes Immunological techniques like (PCR), HPLC and MS and
Non-immunological techniques includes mass spectrometric and chromatographic techniques have also been used. 34

• Anders Ståhlberg et al (2012) studied Reverse transcription and the proximity ligation assay were coupled with
quantitative PCR and used to quantify any combination of DNA, mRNAs,microRNAs (miRNAs), noncoding RNAs
(ncRNAs),and proteins from the same single cell. The method was used on transiently transfected human cells to
determine the intracellular concentrations of plasmids, their transcribed mRNAs, translated proteins, and downstream
RNA targets. 13
• Sandra et al Protein biopharmaceuticals such as monoclonal antibodies and therapeutic proteins are currently in
widespread use for disorders, diabetes and anemia. chromatographic and mass spectrometric tools available to dissect
primary and higher ordethe treatment of various life-threatening diseases including cancer, autoimmune structures, post-
translational modifications, purity and impurity profiles and pharmacokinetic properties of protein therapeutics. 37

13
• Ulrike Leurs et al (2015) described that Protein pharmaceuticals are the fastest growing class of novel
therapeutic The Mass spectrometry has evolved as a powerful tool for the characterization of both primary and
higher order structures of protein pharmaceuticals. 7

• Mahmoud et al (2016) studied biomarkers that are hallmarks of disease has increased demand for high-
performance detection technologies. Implementation of electrochemical methods in clinical analysis may
provide an effective answer to the growing need for rapid, specific, inexpensive and fully-automated means of
biomarker analysis from the past 5 years in the development of electrochemical sensors for clinically-relevant
biomolecules, including small molecules, nucleic acids, and proteins. Various sensing strategies are assessed
according to their potential for reaching relevant limits of sensitivity, specificity, and degrees of multiplexing. 22

• Joanna et al (2017) studied that 2-aminothiazoline-4-carboxylic acid (ATCA) is a hydrogen cyanide

metabolite that has been found to be a reliable biomarker of cyanide poisoning, because of its long-term
stability in biological material. This novel method for ATCA determination in post mortem specimen includes
protein .31

• Rui et al (2017) studied Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) is a
powerful tool for molecular analysis , the conventional organic matrices (CHCA, DHB, etc.) used in assisting
analyte ionization suffer from intensive background noise in the mass region below m/z 700, which hinders
MALDI MS applications for small-molecule detection. 20
• Joanna et al studied that 2-aminothiazoline-4-carboxylic acid (ATCA) is a hydrogen cyanide metabolite that
has been found to be a reliable biomarker of cyanide poisoning, because of its long-term stability in biological
material. This novel method for ATCA determination in post mortem specimen includes
protein .31

14
• Harleen Kaur ,et al (2018) demonstrated the analytical techniques have evolved from a work-intensive,
low sensitivity and high volume of reagent and sample consumption endeavor to automated, better
selectivity, lower limit of quantification and cost effective techniques for biological research. The
analytical techniques discussed in this paper includes surface Plasmon resonance, electrophoresis, enzyme
linked immunosorbent assay, Western blotting, flow cytometry, fluorescence activated cell sorting, mass
spectrometry, nuclear magnetic resonance and x-ray crystallography. 8
• Jialing et al studied on development of an automated and biocompatible handheld mass spectrometry
device for rapid and nondestructive diagnosis of human cancer tissues. The device, named MasSpec Pen,
enables controlled and automated delivery of a discrete water droplet to a tissue surface for efficient
extraction of biomolecules. We used the MasSpec Pen for ex vivo molecular analysis of 20 human cancer
thin tissue sections and 253 human patient tissue samples including normal and cancerous tissues from
breast, lung, thyroid, and ovary. The mass spectra obtained presented rich molecular profiles characterized
by a variety of potential cancer biomarkers identified as metabolites, lipids, and proteins. 18
• Rajesh et al studied on Plant-mediated synthesis of metal nanoparticles has been developed as a
substitute to defeat the limitations of conventional synthesis approaches such as physical and chemical
methods. Biomolecules, such as proteins, amino acids, enzymes, flavonoids, and terpenoids from several
plant extracts have been used as a stabilising and reducing agents for the synthesis of AgNPs. 19

15
 FOR CARBOHYDRATES
• Manns et al (2014) studied The monosaccharide composition of four different samples of brown seaweeds Laminaria digitata and
Saccharina latissima were compared by different high performance anion exchange chromatography (HPAEC) methods after
different acid hydrolysis treatments or a cellulase treatment. A two-step treatment of 72% (w/w) H2SO4 + 4% (w/w) H2SO4
performed best, but cellulase treatment released more glucose than acid treatments. HPAEC with pulsed amperometric detection
(PAD) allowed quantification of all present neutral sugars and the sugar alcohol mannitol. 33

• Both et al (2014)studied Mass Spectrometry (MS) is the primary analytical technique used to characterise the complex
oligosaccharide structures which decorate cell surfaces. The relatively limited number of monomeric glycan building blocks found
in mammals can be assembled in numerous ways to produce a diverse set of higher-order structures. A number of these
monosaccharide units are simple epimers of one another and when combined produce diastereomers of glycoconjugates that cannot
be distinguished by existing MS-based techniques. 35

• Synytsya et al (2014) studied that Glucans are most widespread polysaccharides in the nature. There is a large diversity in
their molecular weight and configuration depending on the original source. According to the anomeric structure of
glucoseunits it is possible to distinguish linear and branched α-, β- as well as mixed α,β-glucans with various glycoside bond
positions and molecular masses. NMR spectroscopy is known as a powerful tool in structural analysis of glucans both in
solution and in solid state. Vibration spectroscopic methods (FTIR, Raman) are sensitive to anomeric structure of glucans
and can be used for purity control as well. Molecular weight distribution, homogeneity and branching of glucans can be
estimated by size-exclusion chromatography (SEC), laser light scattering (LLS) and viscometry. 38

• Santos et al (2016) studied near infrared spectroscopy is proposed to predict two of the most relevant coffee
parameters during the roasting process, sucrose and colour., the methodology was developed taking in consideration
different coffee varieties (Arabica and Robusta), coffee origins (Brazil, East-Timor, India and Uganda) and roasting
process procedures (slow and fast). All near infrared spectroscopy-based calibrations were developed resorting to
partial least squares regression. The results proved the suitability of this methodology as demonstrated by rangeerror-
ratio and coefficient of determination higher than 10 and 0.85 respectively. 37

16
• Anthimopoulos et al (2015) studied Computer Vision-Based Carbohydrate Estimation for Type 1 Patients With
Diabetes Using Smartphones, theIndividuals with type 1 diabetes (T1D) have to count the carbohydrates (CHOs) of
their meal to estimate the prandial insulin dose needed to compensate for the meal’s effect on blood glucose levels.
CHO counting is very challenging but also crucial, since an error of 20 grams can substantially impair postprandial
control ,the GoCARB system is a smartphone application designed to support T1D patients with CHO counting of
nonpacked foods. In a typical scenario, the user places a reference card next to the dish and acquires 2 images with
his/her smartphone. From these images, the plate is detected and the different food items on the plate are automatically
segmented and recognized, while their 3D shape is reconstructed. Finally, the food volumes are calculated and the
CHO content is estimated by combining the previous results and using the USDA nutritional database. 47
• M´arcio et al proposed a method A method for estimation of sugarcane (Saccharum spp.) biomass crystallinity using
near infrared spectroscopy (NIR) and partial least squares regression (PLS) as an alternative to the standard method
using X-ray diffractometry (XRD) is proposed. Crystallinity was obtained using XRD from sugarcane bagasse. NIR
spectra were obtained of the same material. PLS models were built using the NIR and crystallinity values. Cellulose
crystallinity ranged from 50 to 81%. Two variable selection algorithms were applied to improve the predictive ability of
models, i.e. a) Ordered Predictors Selection (OPS) and b) Genetic Algorithm. The best model, obtained with the OPS
algorithm, presented values of correlation coefficient of prediction, root mean squared error of prediction and ratio of
performance deviation equals to 0.92, 3.01 and 1.71, respectively. A scatter matrix among lignin, cellulose,
hemicellulose, ash and crystallinity was built that showed that there was no correlation among these properties for the
samples studied. 48
• Rhyner et al studied a comparitive study for Carbohydrate Estimation by a Mobile Phone-Based System Versus Self-
Estimations of Individuals With Type 1 Diabetes Mellitus , The study was conducted at the Bern University Hospital,
“Inselspital” (Bern, Switzerland) and involved 19 adult volunteers with type 1 diabetes, each participating once. Each
study day, a total of six meals of broad diversity were taken from the hospital’s restaurant and presented to the
participants. The food items were weighed on a standard balance and the true amount of carbohydrate was calculated
from the USDA nutrient database. Participants were asked to count the carbohydrate content of each meal
independently and then by using GoCARB. At the end of each session, a questionnaire was completed to assess
theuser’s experience with GoCARB. 49

17
 For Lipids

• Robert et al discused the direct determination of lipids including spectrophotometric ( turbiditery ) , creamatocrit , and
enzymatic methods including the isolation and gravimetric methods . 14

• Jialing et al report et al the development of an automated and biocompatible handheld mass spectrometry device for
rapid and non destructive diagnosis of human cancer tissues. The device, named MasSpec Pen, enables controlled and
automated delivery of a discrete water droplet to a tissue surface for efficient extraction of biomolecules. We used the
MasSpec Pen for ex vivo molecular analysis of 20 human cancer thin tissue sections and 253 human patient tissue
samples including normal and cancerous tissues from breast, lung, thyroid, and ovary. The mass spectra obtained
presented rich molecular profiles characterized by a variety of potential cancer biomarkers identified as metabolites,
lipids, and proteins. 21

• Nunes et al Edible oils and fats are one of the foods most frequently counterfeited in many countries. Therefore,
monitoring the authenticity and overall quality of these products is ultimately required. Chemometric analyses, such as
Partial Least Square (PLS), Linear Discriminant Analysis (LDA), Soft Independent Modeling of Class Analogy
(SIMCA), and others, applied to vibrational spectroscopic data have enabled the development of methods useful to assess
quality aspects (authenticity, adulteration, free fatty acids and trans content, iodine, peroxide and saponification values,
and others) of edible fats and oil. 39
• Cajka et al studied Liquid chromatography-mass spectrometry (LC-MS)-based lipidomics has undergone dramatic
developments over the past decadeLCMS- based lipidomics methods involve lipid extraction schemes using
chloroform/MeOH or methyl tert-butyl ether (MTBE)/MeOH, both with addition of internal standards covering each
lipid class; LC separation of lipids using short microbore C18 or C8 columns with sub-2-μm or 2.6–2.8-μm (fusedcore)
particle size with analysis time <30 min; electrospray ionization in positive- and negative-ion modes with full spectra
acquisition using high-resolution MS with capability to MS/MS. 40

18
• Pericas et al discussed that by products of arachidonic (AA) and docosahexaenoic acid (DHA) oxidation are
highly relevant for the study of free radical associated conditions in the perinatal period. A validated liquid
chromatographic method to determine peroxidation lipid biomarkers. Serum samples from severely
depressed newborn infants (Apgarscore 1 min <3; arterial cord pH <7.00). Small sample volume and simple
sample treatment.High throughput of sample analysis and high selectivity for different isoprostanes isomers.
41

• Xiaoxiao et al studied that lipidomics has been significantly advanced by mass spectrometric analysis. The
distinction and quantitation of the unsaturated lipid isomers, however, remain a long-standing challenge. In
this study, we have developed an analytical tool for both identification and quantitation of lipid C=C location
isomers from complex mixtures using online Paternò–Büchi reaction coupled with tandem mass
spectrometry (MS/MS). The potential of this method has been demonstrated with an implementation into
shotgun lipid analysis of animal tissues. Among 96 of the unsaturated fatty acids and glycerophospholipids
identified from rat brain tissue, 50% of them were found as mixtures of C=C location isomers; for the first
time, to our knowledge, the quantitative information of lipid C=C isomers from a broad range of classes was
obtained . 42
• Rolere et al investigated that rapid and non-destructive method was developed of lipids (ester and
carboxyl groups) and proteins (amides).to investigate some specific functional groups contained in lipids
(ester and carboxyl groups) and proteins (amides). Ester and carboxyl groups were quantified using
calibration curves developed from synthetic cis-1,4-polyisoprene mixtures with either methyl stearate or
stearic acid. 43
• Min Jia et al studied Surface-enhanced Raman spectroscopy (SERS) is a powerful tool for detecting
biomolecules due to its high sensitivity, rapidness and specificity in identifying molecular structures. This
review focuses on the SERS analysis of biomolecules originated from humans, animals, plants and
microorganisms, combined with nanomaterials as SERS substrates and nanotags. Recent advances in SERS
detection of target molecules were summarized with different detection strategies including label-free and
label-mediated types. 11

19
• Mishra et al (2015) Studied successfully employed sulpho-phospho-vanillin (SPV) colorimetric method
for direct quantitative measurement of lipids within liquid microalgal culture. The SPV reacts with lipids to
produce a distinct pink color, and its intensity can be quantified using spectrophotometricmethods by
measuring absorbance at 530 nm. This method was employed for a rapid quantification of intracellular
lipid contents within Chlorella sp., Monoraphidium sp., Ettlia sp. and Nannochloropsis sp., all of which
were found to have lipid contents ranging in between 10% and 30%. Subsequent analysis of the biomass
using gas chromatography confirmed that our protocol is highly accurate (R2 = 0.99). 45

20
 For Nucleic Acid
• Nicholas et al studied Validation of a Real-Time PCR Based on Qualitative Assay for the Detection of
Methylated SEPT9 DNA in Human Plasma. 46

• Ola M. et al A simple and highly sensitive liquid chromatography–tandem mass spectrometry (LC ‐MS/MS)
bioanalytical method was developed and fully validated for the first time for the simultaneous determination of
newly discovered antiviral drugs, namely sofosbuvir (SOF) and daclatasvir (DAC) in human plasma. Tadalafil
(TAD) was used as internal standard (IS). SOF, DAC and TAD (IS) were extracted from plasma using liquid–
liquid extraction technique with methyl tert ‐butyl ether. 23

• Somanath et al developed digital polymerase chain reaction (dPCR) technology allows absolute and accurate
quantifi-cation of nucleic acid target sequences without need for a reference standard. Due to these properties,
this technique has the potential to not only improve routine quantitative nucleic acid analysis, but also to be used
as a reference method for certification of nucleic acid RM. The article focuses on the use and application of both
dPCR and RMs for accurate quantification. 25

• Amin et al described Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most
sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for
more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine
practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Here we
present methods to determine the limit of detection (LoD) and the limit of quantification (LoQ) as applicable to
qPCR. 26

21
• Alexandra et al have demonstrated how digital PCR (dPCR) enables preciseand sensitive quantification of
nucleic acids in a wide range of applications in both healthcare and envi-ronmental analysis. This has occurred
in parallel with the advances in partitioning fluidics that enable areaction to be subdivided into an increasing
number of partitions. As the majority of dPCR systems arebased on detection in two discrete optical channels,
most research to date has focused on quantificationof one or two targets within a single reaction. 27

• Khalid B Beshir et al studied quantitative PCR method was developed in which distinct fluorescent signals are
generated from the human and parasite DNA components in each blood sample. The human amplification target
in this assay is the b tubulin gene, and the parasite target is the unique methionine tRNA gene (pgmet), which
exhibits perfect sequence identity in all six Plasmodium species that naturally infect humans. In a small series of
malaria cases treated as hospital in-patients, the abundance of pgmet DNA was estimated relative to the human
DNA target in daily peripheral blood samples, and parasite clearance times calculated. 12
• Jialing et al studied on development of an automated and biocompatible handheld mass spectrometry device
for rapid and nondestructive diagnosis of human cancer tissues. The device, named MasSpec Pen, enables
controlled and automated delivery of a discrete water droplet to a tissue surface for efficient extraction of
biomolecules. We used the MasSpec Pen for ex vivo molecular analysis of 20 human cancer thin tissue sections
and 253 human patient tissue samples including normal and cancerous tissues from breast, lung, thyroid, and
ovary. The mass spectra obtained presented rich molecular profiles characterized by a variety of potential cancer
biomarkers identified as metabolites, lipids, and proteins. 18

• Cheng et al Oxidative DNA damage plays crucial roles in the pathogenesis of numerous diseases including
cancer. 8-hydroxy-2′-deoxyguanosine (8-OHdG) is the most representative product of oxidative modifications
of DNA, and urinary 8-OHdG is potentially the best non-invasive biomarker of oxidative damage to DNA.
Herein, we developed a sensitive, specific and accurate method for quantification of 8-OHdG in human urine.
The urine samples were pretreated using off-line solid-phase extraction (SPE), followed by ultrahigh
performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. 30

22
• .

23
24
25
• Gautam et al pituitary adenomas secrete distinct pituitary hormones (most often prolactin, growth
hormone, or adrenocorticotropic hormone). While these tumors are histologically benign, they have potent
endocrine effects that lead to significant morbidity and shortened lifespan. Because of their
pathophysiologic endocrine secretion and anatomic location near critical neural/vascular structures,
hormone-secreting pituitary adenomas require defined management paradigms that can include relief of
mass effect and biochemical remission. Management of hormone-secreting pituitary adenomas involves a
multidisciplinary approach that can incorporate surgical, medical, and/or radiation therapies. 32

• Yixiao et al studied HPLC–UV is a highly effective screening method based on a column separation and
217 detection under the maximum UV absorption of analyte.Liquid-liquid extraction (LLE) used for
insulin extraction. Generally, dichloromethane is added to the insulin standard solution or biological
sample . 15

26
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