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A novel assay, Fast parallel proteolysis (FASTpp), might help swift screening of specific stability defects of
specific proteins in individual cancer patients. FASTpp measures the quantity of protein that resists
digestion under various conditions. A thermostable protease is used, which cleaves specifically at
exposed hydrophobic residues.The FASTpp assay combines the thermal unfolding, specificity of a
thermostable protease for the unfolded fraction with the separation power of SDS-PAGE. Due to this
combination, FASTpp can detect changes in the fraction folded over a large physico-chemical range of
conditions including temperatures up to 85C, pH 6-9, presence or absence of the whole cytosolic proteome.
Specific diseases caused by insertions/deletions Tay-Sachs Disease. Tay-Sachs Disease is a fatal disease
affecting the central nervous system. Symptoms do not appear until approximately 6 months of age. The
child becomes blind, deaf, unable to swallow, atrophied, and paralytic. Mutations in the -hexosaminidase A
(Hex A) gene are known to affect the onset of Tay-Sachs. Cancer Insertion/deletion mutations cause
colorectal cancer and other cancers with microsatellite instability. While environmental factors contribute
to the progression of prostate cancer, genetic component also will. There are over 500 mutations on
chromosome 17 that seem to play a role in the development of breast and ovarian cancer in the BRCA1 gene,
many of which are Insertion/deletion.
SNP and DISEASE One study even identified two genes in which particular variants can slow the onset of
AIDS, demonstrating the potential of this approach for understanding why people vary in their susceptibility
to infectious diseases. New technologies that are slashing the costs of sequencing and genome analyses will
make possible the simultaneous genome-wide search for SNPs and other DNA alterations in individuals.
Proteomics Proteomics is the large-scale study of proteins, particularly their structures and functions.
Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic
pathways of cells. The proteome consists of the entire complement of proteins, including the modifications
made to a particular set of proteins, produced by an organism or system. This will vary with time and distinct
requirements, or stresses, that a cell or organism undergoes.
Number of Proteins in Human Analyzing genome sequences alone will not lead to new therapies to fight
human diseases. The human genome has approximately 35,000 genes and theoretically the ability to encode
up to 35,000 corresponding proteins. The occurrence of alternative RNA splicing and PTM, such as
phosphorylations, acetylations, and glycosylations, or protein cleavages may increase the expression of
proteins to 500,0001,000,000. The proteins reflect more accurately the intrinsic genetic mechanisms of the
cell and their impact on the microenvironment, as they are the effectors and characterize
Proteomics in Biomedical Research Biomarkers are biomolecules that is associated with an increased risk of
the disease and serve as indicators of biological and pathological processes or physiological and
pharmacological responses to a drug. Proteins that are impt indicators of physiological or pathological states
may contribute to the early diagnosis of disease, which may provide a basis for identifying the underlying
mechanism of disease development. These differentially expressed proteins in serum have become an impt
in monitoring the state for disease. Comprehensive proteome of human serum fluid with high accuracy and
availability has the potential to open new doors for disease biomarker discovery and for disease diagnostics.
Proteomics in Cancer Diagnostics Allied to genomics, proteomics technologies is valuable for identifying
new markers that improve screening, early diagnosis, prognosis and prediction of therapeutic response or
toxicity, as well as the identification of new therapeutic targets. Studies on the proteome in cancer have
used tissue samples and biological fluids including serum, plasma, saliva, and cerebrospinal fluid in search for
the detection of diagnostic, predictive, and prognostic biomarkers. Among the proteomics tools, mass
spectrometry (MS) is one of the most used techniques for identifying unknown proteins. The mass
spectrometer is an analytic instrument capable of converting neutral molecules into gaseous ions and
separating them according to their mass-to-charge (m/z) ratio by using an electromagnetic field.
Tandem mass spectrometry (MS) offers info about specific ions. In this approach, distinct ions are selected
based on their m/z from the first round of MS and are fragmented by a number of methods of dissociation,
such as colliding the ions with a stream of inert gas, as in collision-induced dissociation or higher energy
collision dissociation. Other methods of ion fragmentation include electron-transfer dissociation and electroncapture dissociation .
These fragments are then separated based on their individual m/z ratios in another round of MS. MS/MS is
commonly used to sequence proteins and oligonucleotides, as the fragments can be used to match predicted
peptide or nucleic acid sequences that are found in databases. These sequence fragments can then be
organized in silico into full-length sequence predictions.
A sample is injected into the MS, ionized and accelerated and then analyzed by MS1. Ions from the MS1
spectra are then selectively fragmented and analyzed by MS2 to give the spectra for the ion fragments.
Sugars, AA and nucleotides can polymerize to form macromolecules called polysaccharides, proteins and
nucleic acids. Sugars, AA and nucleotides polymerize to release water. In hydrolysis, a water molecule reacts
with the bond linking the monomers. A monomer is broken off, resulting in a shorter polymer. Sugars are
defined by the presence of an carbonyl group and multiple hydroxyl groups. Sugars like glucose can exist in
both linear and ring forms. Like many organic molecules, sugars are chiral molecules- they can exist as righthanded (D) or left handed (L) isomers. Right-handed (D) forms predominate in cells.
When glucose forms a ring, the hydroxyl group
attached to the number 1 carbon is locked into one of
two alternate positions: either below the plane of the
ring, or above it. These two ring forms of glucose are
called alpha () (down) and beta () (up), respectively:
Examples of sugar polymers: Starch is polymerized
glucose, in which -glucose monomers are polymerized via a 1-4
linkage. Cellulose, on the other hand, is polymerized glucose, in
which -glucose monomers are polymerized via a 1-4 linkage.
(Animals dont have enzymes to catalyze the hydrolysis of the
-glycosidic link in cellulose!)
Starch Structure: Starch is made from chains of -glucose
molecules. These are linked by glycosidic bonds. Starch is found in many parts of a plant as starch grains.
Why is starch a good molecule for storage in plants?
It is insoluble, so doesnt draw water into cells by osmosis. Wont easily diffuse out of cells because it is
insoluble, It can be stored in a small space because the tight coils make it compact, Can be easily hydrolyzed
to give -glucose , which can be used in respiration, They are a reserve form of sugar for times when free
sugar In diet is absent. Starches called amylose(an unbranched -glucose polymer) and pectin(a branched
polymer) are the storage polysaccharides found in plants.
Significance of StarchGreen plants use starch as their energy store. An exception is the family Asteraceae,
where starch is replace by fructan inulin. Photosynthesis, plants use light energy to produce glucose from CO2.
Starch The glucose is stored mainly in the form of starch granules, in plastids such as chloroplasts and
especially amyloplasts. Toward the end of the growing season, starch accumulates in twigs of trees near the
buds. Fruit, seeds, rhizomes, and tubers store starch to prepare for the next growing season.
From Glucose to Starch Glucose is soluble in water, binds with water and then takes up much
space and is osmotically active. Glucose in the form of starch, is not soluble, therefore osmotically
inactive and can be stored much more compactly.Glucose molecules are bound in starch by the
easily hydrolyzed alpha bonds. The same type of bond is found in the animal reserve
polysaccharide glycogen. This is in contrast to many structural polysaccharides such as chitin,
cellulose and peptidoglycan, which are bound by beta bonds and are much more resistant to
hydrolysis.
Production of glucose 6-phosphate Glucose 6-phosphate is produced by phosphorylation of
glucose on the sixth carbon. This is catalyzed by the enzyme hexokinase in most cells, and, in
higher animals, glucokinase in certain cells, most notably liver cells. One molecule of ATP is
consumed in this reaction. The reason for the immediate phosphorylation of glucose is to
prevent diffusion out of the cell. The phosphorylation add a charged phosphate grp so the glucose
6-phosphate cannot easily cross the cell membrane.
Two Forms of Starch
Around 30%, tightly
packed structure,
more resistant to
Digestion. Amylose can
Exist in Helical Forms.
Around 70%,highly
branched structure,
being formed of 2,000
to 200,000 glucose
units can be quickly
degraded
Amylopectin on the other hand is a branched-chain polysaccharide where in addition to the -1,4glycosidic bonds there is the occasional -1,6-glycosidic bonds. Branching occurs about every 2430 glucose units. Helical structure of amylopectin is disrupted by branching.
Glycogen A multibranched polysaccharide of glucose that is a form of energy storage in animals
and fungi. In humans, glycogen is made and stored in the cells of the liver and the muscles, and
functions as the secondary longterm energy storage (primary energy stores being fats).Glycogen
is the analogue of starch, having a similar structure to amylopectin, but more branched and
compact. Glycogen is found in the form of granules in the cytoplasm in many cell types, and plays
an impt role in the glucose cycle. Glycogen forms an energy reserve that can be quickly mobilized
to meet the need for glucose, but is less compact than the energy reserves of triglycerides.
Glycogen is a branched biopolymer consisting of linear chains of glucose residues with further
chains branching off every 10 glucoses. Glucoses are linked together linearly by (14) glycosidic
bonds. Branches are linked to the chains and are branched off by (16) glycosidic bonds.
Cellulose Made of -glucose. To form glycosidic links, each -glucose molecule is rotated 180o
compared to the one next to it. Has straight, unbranched chains that run parallel to one another.
Hydrogen bond links the chains. The -glycosidic link between glucose molecules in cellulose
results in a polymer that forms a long linear strand. The hydroxyl groups of one cellulose molecule
are free to H bond with the hydroxyls of adjacent molecules. In plants, the long strands of cellulose
bundle together to form microfibrils. Bundles of microfibrils form plant cell walls.
So many hydrogen bonds help to strength cellulose
This makes cellulose a good structural material, hence its use in plant cell walls to aid rigidity
cellulose does this by grouping together to form microfibrils
Cellulose prevents cell bursting, so they are turgid when full with water. This helps support stems
Other impt structural polysaccharides are chitin and peptidoglycan Both composed of polymers of
amino sugars, such as N-acetyl-glucosamine (chitin) or [N-acetyl-glucosamine plus N-acetylmuramic acid] (Peptidoglycan). A mesh of peptidoglycan chains, crosslinked by covalent bonds,
make up the tough and flexible bacterial cell wall. (antibiotics poison the bacterial enzymes that
synthesize cell wall)
Chitin (mono monomer) (Parallel strands joined by hydrogen bonds) Chitin is a long-chain
polymer of a Nacetylglucosamine, a derivative of glucose. The main component of the cell walls
of fungi, the exoskeleton of arthropods such as and insects, the radulae of molluscs, and the beaks
and internal shells of cephalopods. The structure of chitin is comparable to the polysaccharide
cellulose, forming crystalline nanofibrils. In terms of function, it may be compared to the protein
keratin. It form covalent -1,4 linkages (similar linkages between glucose units forming cellulose).
Chitin is cellulose with one hydroxyl group replaced with an acetyl amine group.
Peptidoglycan (Parallel strands joined by peptide bonds) also known as murein, is a polymer
consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of
most bacteria, forming the cell wall. The sugar component consists of alternating residues of (1,4) linked N-acetylglucosamine and N-acetylmuramic acid. Attached to the N-acetylmuramic acid
is a peptide chain of three to five amino acids. The peptide chain can be crosslinked to the peptide
chain of another strand forming the 3D mesh-like layer. Peptidoglycan serves a structural role in
the bacterial cell wall, giving structural strength, as well as counteracting the osmotic pressure of
the cytoplasm. peptidoglycan helps maintain the structural strength of the cell. Peptidoglycan is
also involved in binary fission during bacterial cell reproduction. The peptidoglycan layer is
substantially thicker in Gram-positive bacteria than in Gram-negative bacteria, with the
attachment of the S-layer. Peptidoglycan forms around 90% of the dry weight of Gram-positive
bacteria but only 10% of Gram-negative strains.
Meso-diaminopimelic acid (DAP) for Gram Positive
Nucleic Acids
Two classes of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)
Cells use DNA to determine and control the synthesis of proteins with the help of mRNA.
mRNA dictates the synthesis of protein from amino acids delivered by transfer RNA.
Made up from three components: nucleobases, sugars and phosphoric acid.
If U were used in DNA, then when the C in a G:C base pair
deaminated to become U, the G:C base pair would become
a G:U base pair. A G:U base pair is detected by the ongoing
DNA-repair enzymes. Since U is not used in DNA, any U
formed can be recognized as illegitimate and have to come
from mutated a C; it is cut out by repair enzymes and replaced
with C.
The nucleotide (Base + sugar + phosphate)
Note: to distinguish between sugar and base, positions in the
sugar are designated with a prime ( )
A strand of DNA is made by attaching one nucleotide onto a
second one, and then a third one on the second one, etc. DNA
is synthesize in beginning at the 5 end and progressing towards
the 3 end. Consequently, the convention when writing out the
nucleotide sequence of a nucleic acid is to begin with the 5 nucleotide on the left and end with
the 3 nucleotide on the right
+
+ 2 + 3
One of the impt functions of proteins is to serve as catalysts for chemical reactions necessary for life. Proteins that
+
functions as chemical catalysts are called enzymes. The complex surface of a folded protein creates crevices that can
The position of the equilibrium is measured by the equilibrium constant = 3
2
bind other molecules. The interior of these crevices is lined with the chemically reactive side chains of the various AA.
Now, in dilute solutions of acid, 3 + stays roughly constant at about 56 mol/dm3. we therefore Consequently, proteins are excellent and very specific chemical catalysts. By stablilizing the transition state of the
+
reaction, enzymes lower the activation energy.
define a new equilibrium constant the acidity constant = 3
Synthesis of proteins
This is also expressed in logarithmic form are as follows: = 10
Transcription Translation Post-translational modification: phosphorylation, acetylation, methylation, glycosylation
Because of the minus sign, the lower the pKa the higher the Ka and the stronger the acid.
Post-Translational Modifications
It turns out that the pKa of an acid is the pH at which it is exactly half-dissociated. This can be
Proteins are involved in cellular signaling and metabolic regulation. They are subject to biological modifications. Almost all
shown by re-arranging the expression for Ka:
protein sequences are post-translationally modified and 200 types of modifications of amino acid residues are known.
+
3 =
The dynamic nature of the proteome
The proteome of the cell is changing. Various extra-cellular, and other signals activate pathways of proteins. A key
mechanism of protein activation is post-translational modification These pathways may lead to other genes being
=
equation: = =
6r
When an electric field is applied, a flow of solvent is generated inside the capillary, called electro-osmotic
flow. The velocity of the electro-osmotic flow depends on the electro-osmotic mobility(eo) which in turn
depends on the charge density on the capillary internal wall and the buffer characteristics. The electroosmotic velocity (Veo) is given by: = =
(+)
Theoretical plate: After introduction of the sample, each analyte ion of the sample migrates within
the background electrolyte as an independent zone, according to its electrophoretic mobility. Zone
dispersion, that is the spreading of each solute band, results from different phenomena. Under
ideal conditions the sole contribution to the solute-zone broadening is molecular diffusion of the
solute. In ideal case the efficiency of the zone, expressed as the number of theoretical plates (N):
(+)
=
Plates do not really exist. They serve as a way of measuring column
2
efficiency, either by stating the number of theoretical plates in a column, N (the more plates the
better). Other phenomena such as heat dissipation, sample adsorption onto the capillary wall,
mismatched conductivity between sample and buffer, length of the injection plug, detector cell
size and unlevelled buffer reservoirs can also significantly contribute to band dispersion (N
becomes smaller). Separation between 2 bands, a and b (expressed as the resolution, Rs) can be
obtained by modifying the electrophoretic mobility of the analytes, the electro-osmotic mobility
induced in the capillary and by increasing the efficiency for the band of each analyte, according to
the equation: =
()
4( +)
Immobilized pH gradients (IPG) Produced by incorporating substances called immobilines in the gel Useful characteristics of antibodies They are specific to the substances used to generate them. They are
polymerization process. Immobilines are not zwitterionic; they are either acidic or basic. The pH
immunogenic themselves (i.e. it is possible to raise antibodies to antibodies). The FC portion can be modified without
depends on the ration of these immobilines. Controlled mixing during gel casting is required to obtain affecting the specificity or affinity of binding. They bind the AG with high affinity (makes the assay more sensitive).
a good pH gradient. Once cast, these gels can be prepared in quantity, dried, and stored, so that a
Antigen An AG is a molecule capable of inducing an immune response when entering the body. Two classes of AG can
rehydration step just prior to use with reproducible gradients.
be distinguished: complete and incomplete AG. Complete AG can induces an immune response by themselves.
Properties of IPG Acrylamido buffers are an alternative means to form pH gradients that circumvent Incomplete AG , also called hapten, has to attach to protein carries to trigger the production of antibodies. The binding
most of the limitations of carrier ampholytes. Chemically, they are acrylamide derivatives of simple site of the AG, the epitope, makes up a small area of the total AG structure. Epitopes can be continuous or discontinuous.
buffers and do not exhibit amphoteric behavior. The acrylic function of an acrylamido buffer
Ab-Ag Complex The complex formation is reversible and depends on the interplay of several forces. Electrostatic
copolymerizes with the gel matrix. By pouring a gel that incorporates an appropriate gradient
interaction between the positively charged amino group and negatively charged carboxyl group. H bonds between
of acrylamido buffers, an immobilized pH gradient (IPG) is formed. Creating an immobilized pH
hydroxyl, amino and carboxyl groups Van der Waals-forces The binding strength between a single epitope and
gradient. (A, B, C) A gradient of acrylamido buffers in an acrylamide solution is cast into a slab gel that paratope is referred to as their affinity, which can be quantified by the equilibrium constant (Keq): Keq=[Ab-Ag]/[Ab][Ag]
is crosslinked to a plastic support film. (D) The gel is washed to remove polymerization byproducts. Immunoassay Formats Limited (competitive) or excess reagents (non-competitive) Homogenous or heterogeneous
(E) The gel is dried for storage. (F) The pH at any point in the gel is determined by the mixture of
Labelled or unlabelled
buffers crosslinked into the gel at that site.
Limited reagent Immunoassay (Competitive) The AG molecules with the sample compete with a fixed amount of labelled
Advantages of IPG The protein sample can be applied immediately. The pH gradient is stable and
AG for the limited amount of AB binding sites. Only a fraction of the ABbinding sites are bound with labeled AG.
does not drift in an electric field. Additionally, the gels are not susceptible to cathodic drift, because Excess reagent Immunoassay (Non-Competitive) The AG sample is added to an excess of AB reagent leading to fractional
the buffers that form the pH gradient are immobilized within the gel matrix. Individual Immobiline
occupancy of AB binding sites. Secondary AB with a label is added andsandwich complex is formed allowing detection.
species with a specific pK value are available, suitable for casting gradients from pH 310. Because
Home Pregnancy Test Detect the Glycoprotein hormone human chorionic gonadotropin (hCG) A few days after
reproducible linear gradients with a slope as low as 0.01 pH units/cm can separate proteins with pI
conception, hCG appears in the urine and its concentration increase rapidly during the first week of pregnancy. The
differences of 0.001 pH units. The resolution possible with immobilized pH gradient gels is 10100
strip component is composed of an adsorbent material. Once the urine sample is applied, the liquid moves along the
times greater than that obtained with carrier ampholytebased IEF. IEF is best performed in a flatbed strip by capillary action and the assay-reactions are carried out in flow.
electrophoresis apparatus. This type of apparatus allows very effective cooling, which is necessary due Enzyme-linked immunosorbent assays (ELISA) Enzymes are labels Reaction of enzyme with colorless substrate
to the high voltages. A variety of precast gels for IEF, including ready to use carrier ampholyte gels,
produces colored product. Indirect ELISA detects antibodies in serum Sandwich ELISA used to detect AG
dried IPG gels, and dried acrylamide gels. These gels are ready for reswelling in a mixture of carrier
Molecular Recognition-Biosensor Biosensor- is an analytical device that combines a biological sensing element (such as
ampholytes and any other additives desired, such as detergent and denaturants.
an AB, enzyme or whole cell) with a transducer to produce a signal proportional to the analyte concentration.
2D Gel Electrophoresis Two modes of electrophoresis are combined on a single gel. Usually,
Biosensors signal is originated from a change in proton concentration, release or uptake of gases, light emission,
proteins are separated by IEF in 1 dimension, based on pI. Followed by SDS-PAGE in a perpendicular absorption and so forth, The response is brought about by the reaction of biomolecule and target compound. The
direction, based on size. Mixtures of thousands of proteins can be separated. The result can be
transducer converts this signal into a measurable response such as current, potential or absorption of light through
compared to electronic databases. This method resolves few 1000 protein spots.
electrochemical or optical means. This signal can be further amplified, processed and stored for later analysis.
Capillary Electrophoresis (CE) Separation method carried out in a buffer-filled capillary tube that is
The enzyme is immobilized on a platinum electrode, and covered with a thin polyurethane (outer) membrane to protect
typically 20 to 100 m in internal diameter and 10 to 100 cm in length. The tube extends between
the enzyme layer, and reduce the dependence of the sensor on blood oxygen levels. Glucose oxidase, in its oxidized form,
two buffer reservoirs that also hold Pt electrodes. The sample is introduced into one end of the
oxidises glucose entering the sensor to gluconic acid; resulting in the conversion of the enzyme to its reduced form. The
tubing, and a dc potential in the 10 to 30 kV range is applied between the two electrodes throughout enzyme does not remain in this form for long.
the separation. The separated analytes are observed by a detector at the end of the capillary opposite Definitions Biological receptor: A macromolecule / cell / tissue that recognizes the target analyte Transducer: Device that
the end where the sample was introduced. Charged analytes migrate in the presence of an electric
converts the biological recognition event into a measurable signal Processor: Converts the measured signal into a signal
field. Separation is based on differential rates of migration.
that can be interpreted by the user, e.g., a number, a colour, a meter readout.
Hydrodynamic injection can be performed in: Pressure injection or vacuum injection, gravity flow
Analytes Microbes: E. coli, etc. Small molecules: glucose, CO2, amino acids Bio-macromolecules: DNA, RNA,
injection. The anodic end is removed from the buffer reservoir and placed in the sample solution. The enzymes, proteins, hormones, viruses Ways to obtain analyte sample: 1. Non-Contacting: Electromagnetic radiation
capillary end is then raised so that the liquid level in the sample vial is at a height h above the level of
(IR/UV/Visible light, usually requires a probe) Taking a gas sample near surface Does not interfere with the subject
the cathodic buffer, and is held in this position for a fixed time t.
2. Contacting: Invasive/Non-invasive Can evoke a response/ change analyte 3. Removing: More or less traumatic
Electrokinetic injection involve drawing sample ions into the capillary interior with an applied potential. (blood versus urine) Toxic probe molecules or other additives (e.g. heparin) can be added Most commonly used
A high voltage is applied over the capillary between the sample vial and the destination vial for a given Bio receptors: Biological interactions Weak, non-covalent interactions Spontaneous (self-assembly) Highly specific
time. This causes the sample to move into the capillary according to its apparent mobility, app.
(single atom differences) Complementary (lock-key) Enzymes/ substrates Most commonly used biological receptors.
Problem: discrimination occurs between different components in the sample.
They are catalysts. Coupled enzyme reactions to give coloured reaction product or emitted photons. AB/ AG Highly
Order of Elution with EOF: small cations, large cations, neutral, large anions, small anions.
selective interactions and very tight binding. AB can be raised against almost any antigen. Usually needs to be linked to
Capillary Electrophoresis Separation based on electrophoretic mobility Primary applications in
other probe for detection. Receptor proteins Many receptor proteins on surface of cells and embedded into
bioanalysis DNA sequencing DNA fragment analysis Multiple modes for improved selectivity of
membranes. Applications mainly expected in detection of neuro-transmitters, hormones, neuro-active. Highly selective
neutrals -- Capillary Zone electrophoresis (CZE) -- Micellar electrokinetic chromatography (MEKC)
but often difficult to isolate. Use of intact biomembranes or cells. Nucleic acids DNA, RNA diagnostic sensors in chip
DNA capillary electrophoresis During capillary electrophoresis, the products of the cycle sequencing
format; used to detect genetic disorders and expression levels of proteins in parallel. DNA and RNA can be synthesised in
reaction are injected electrokinetically into capillaries. High voltage is applied so that the negatively
the lab. Microorganisms E.g. genetically modified bacterial cells that light up when toxins are presented.
charged DNA fragments move through the polymer in the capillaries toward the + electrode.
Immobilization and surfaces Bio-receptor molecule has to be immobilized on or near the surface of the transducer. The
Detection Options UV-absorption detection Most common mode M limit of detection
immobilization is done either by physical entrapment (e.g. using a membrane) or chemical attachment. Biological
Peptide =210nm, protein and DNA at 260 or 280nm Laser-induced Fluorescence Very sensitive recognition capability should not be lost! Chemical methods of bioreceptor immobilization involve the formation of
Limited to fluorescent species - Mass Spectrometry
covalent bonds, including covalent binding, e.g., cross-linking. Physical methods of bioreceptor immobilization, such as
Modes of CE (CZE) (MEKC) Separates compounds with micelles Capillary Gel Electrophoresis adsorption and entrapment.
Size exclusion using sieving gels Capillary Isoelectric Focusing
Signal transducers The transducer converts the recognition event into a measurable signal. The transducer can take
CZE: + ions move faster than the ions. Relative to the EOF, the + ions moved ahead but ions moved many forms depending upon the parameters being measured. Electrochemical, optical, mass and thermal changes are
backwards. Solution is electrically neutral since anions and cations are surrounded by buffer
the most common. Oxygen sensor: [O2] e current pH sensor (gluconic acid): pH voltage Peroxide sensor: [H2O2]
counterions. Buffer systems dont interfere with the detection of analytes.
e current Coupled to peroxidase rxn: [Dye] colour change Thermal: enthalpy T
Principles of MKEC In MEKC, separation takes place in an electrolyte solution which contains a
Electrochemical Potentiometric -- detect changes in potential at constant current (usually zero). Amperometric -surfactant at a concentration above the critical micellar concentration (cmc). The solute molecules are detect changes in current at constant potential. (currents generated when electrons are exchanged between a biological
distributed between the aqueous buffer and the pseudo-stationary phase composed of micelles,
system and an electrode) Conductometric-- detect changes in conductivity between two electrodes. Piezoelectric
according to the partition coefficient of the solute. The technique can therefore be considered as a crystals: Piezoelectric--These devices detect changes in mass. Micromechanical systems: Cantilever transducers
hybrid of electrophoresis and chromatography. It is a technique that can be used for the separation Electrochemical Transducers Potentiometric Measuring cell potential at (near) zero current. The cell potential is
of neutral and charged solutes, maintaining the efficiency, speed and instrumental suitability of
proportional to the ion concentration. Mainly used to quantify inorganic ions, including H+. In biosensors usually enzyme
capillary electrophoresis. Widely used surfactants in MEKC is the anionic surfactant sodium dodecyl reactions that involve a significant pH change: penicillinase, urease, esterase. Need a reference electrode (e.g. Ag/AgCl)
sulphate, although other surfactants, for ex. cationic surfactants such as etyltrimethylammonium salts. Electrochemical Transducers Amperometric A constant potential applied between a working and a reference
Mechanism At neutral and alkaline pH, a strong electro-osmotic flow is generated and moves the
electrode; the current through the cell is measured continuously. When the oxidation potential of a molecule is
separation buffer ions in the direction of the cathode. If sodium dodecyl sulphate is employed as the reached, it is oxidised and electrons are produced: measured as a current through the cell. Current is linearly related to
surfactant, the electrophoretic migration of the anionic micelle is in the opposite direction, towards the the concentration of the oxidised molecule. Most commonly used in reactions involving REDOX enzymes, e.g., Glucose
anode. As a result, the overall micelle migration velocity is slowed down compared to the bulk flow of oxidase, cholesterol oxidase, etc.
the electrolytic solution. In the case of neutral solutes, since the analyte can partition between the The Glucose Amperometric Biosensor Why is needed? Diabetes A chronic medical condition associated with
micelle and the aqueous buffer, and has no electrophoretic mobility. The analyte migration velocity abnormally high levels of sugar (glucose) in the blood known as hyperglycemia. Normally, blood glucose levels are
will depend only on the partition coefficient between the micelle and the aqueous buffer.
tightly controlled by insulin, a hormone produced by the pancreas which promotes cellular uptake. Type I (IDDM)
Result In the electropherogram, the peaks corresponding to each uncharged solute are always
Pancreas undergoes autoimmune attack by the body itself. Destruction of beta cells thus renders one incapable of
between that of the electro-osmotic flow marker and that of the micelle (the time elapsed between
making insulin. (10%) Type II (NIDDM) Cells exhibit a lack of sensitivity to insulin, thus the pancreas inadequately
these two peaks is called the separation window). For electrically charged solutes, the migration
produces larger than normal quantities in an attempt to increase cellular recognition. (90%) Diabetes leads to diseases
velocity depends on both the partition coefficient of the solute between the micelle and the aqueous such as heart disease and obesity.
buffer, and on the electrophoretic mobility of the solute in the absence of micelle.
The Glucose Biosensor: How is the disease managed? Home blood sugar (glucose) testing is an important part of
Summary of blotting techniques Southern - Restricted DNA on gel denature and trf to filter
controlling blood sugar. One important goal of diabetes treatment is to keep the blood glucose levels near the normal
hybridize with probe = labelled DNA Northern -- RNA on gel transfer to filter hybridize with
range of 70 to 120 mg/dl before meals and under 140 mg/dl at 2 hours after eating.
probe = labelled DNA Western -- protein on gel transfer to filter react with probe(fluroscence, Conductometric Measuring conductance/resistance in a solution. Useful when charges are produced during
hemiluminescence, colorimetric detection) = antibody. Use GE to separate the target Use
enzymatic conversion. Ex: detection of urea (in urine) using urease: Urea + 2H2O Urease 2NH4 + 2HCO3
sequence-specific or shape-specific molecular recognition between probe-target Label the probe and Electrochemical transducers Advantages: Easy to use, Direct interface with electronic displays, Possibility to miniaturize
detect the target
(faster response times) Disadvantages: Limited selectivity, Only works when biological reaction involves electron transfer
Molecular Recognition-Bioassay Most importantly immunoassay, is an analytical method using
Optical transducers Photometric behaviour that can be exploited in biosensors: UV/visible absorption Fluorescence
antibody as reagents to quantitate specific antigen. rely on the highly specific reaction between AB (and phosphorescence) emission Bio-luminiscence Chemi-luminiscence Internal Reflection Spectroscopy (IRS)
and AG. Very sensitive used in bioanalytical chemistry, especially for diagnosis and management of Light scattering methods 400-700nm(UV Infrared) Devices: photomultipliers (convert photons to current),
diseases, pregnancy test and anthrax
spectrophotomers, optic fibres, etc. Advantages: Easy to use Can respond simultaneously to different reactants Can
Bioassays AB and AG have recognition sites, called paratope and epitope respectively. Epitope - a be very accurate, especially when more wavelengths are used Can use optical fibres for efficient photon transport
molecular region on the surface of an antigen capable of eliciting an immune response and of
Very high sensitivity (bio-luminiscence) Disadvantages: Depend on availability of reactant that changes optical
combining with the specific antibody produced by such a response antigenic determinant When
properties Dynamic range only around 102 (where Beer/Lambert law applies) Difficult to miniaturize Response time
paratope and epitope match with each other, Ab-Ag complex is formed. This kind of binding has very may be slow: analyte diffusion Background light interference
high affinity. That explains the high sensitivity and low limits of detection obtained with bioassays. Thermal Transducers Making/breaking chemical bonds in enzymatic reactions results in enthalpy changes. In addition
To detect the assay product, it usually labels either the AB or AG with fluorescent, luminescent,
heats of solution change especially with formation of charged species (e.g. protons). Typically 10-3 K of temperature
radioactive, an enzyme or an electrochemically active group. It can be performed in a large variety
change, can be detected. Reaction of interest may be coupled to reaction that produces more heat (e.g. glucose
formats, in solution or on a solid support, with limited reagent or an excess of reagent.
oxidase coupled to catalase). Advantages: They work for most reactions Disadvantages: Non-selective, Low sensitivity,
Antibody AB is a protein complex used by the immune system to identify and neutralize bacteria and Difficult to miniaturize
viruses. Each AB recognizes a specific AG unique to its target. Produced in living organisms via
Piezo-electric transducers (Mass) The frequency (f) of crystal oscillation depends on the mass (m) of the crystal and
immune response, in response to immunogen. AB, also referred to as immunoglobulins (Ig), consist that of any material adsorbed to its surface (A). Natural oscillation frequency decreases with adsorbed mass according
of four subunits: two identical light chains (~25KDa) and two identical heavy chains (~50KDa) These to the Sauerbrey equation: f = -2.3 x 106 f2m/A Piezo electric materials: Quartz (SiO2 crystals), ceramic materials,
subunits are associated via disulfide bonds and non-covalent interactions to form a Y-shaped
organic polymers.
symmetric dimer. Five types of Ig determined by five types of heavy chains: IgA, IgD, IgE, IgG (most
common AB), IgM Two types of AB can be distinguished: monoclonal AB and polyclonal AB.
Polyclonal AB are isolated from serum. The resulting antiserum will contain a mixture of AB that bind
to different epitopes of AG. This may result in significant cross-reactivities, or interferences, when
employed in immunoassays. Monoclonal AB are a homogeneous population of identical AB
molecules, having identical paratopes and affinity for a single antigenic epitope.
Structure of AB Have the same basic structure. 4 polypeptide chains linked by disulfide bonds. Two
light chains 2 heavy chains AB have two AG binding regions. A hinge region which confers
flexibility on the Molecule A model of an ig molecule. The heavy chains are coloured dark red and
dark blueThe light chain are light red and light blueC mean crystallizable and ab mean AG binding