CHEM4400

Advanced Analytical Chemistry

Labeling Methods
Bo Zheng
bozheng@cuhk.edu.hk
Science Center Rm. 165
 Purpose of Labeling
Labeling allows us:

(1) to detect the molecule.

(2) to perform quantitative measurement. For example, to estimate

the number of salmon fish in a pond, a few individual salmon are
captured, tagged (i.e., labeled) and reinserted into the pond. After a
few days, a number of fish (i.e., the sample) are removed from the
pond, and using the fraction of tagged salmon in this sample, the
entire population can be deducted.
For instance, if the number of salmon initially labeled was 500, and a
few days later 10 of them are found in a group of 200 fish removed
from the pond, then the total number of salmon will be 10,000.

(3) to amplify the detection signal. For example, an enzyme label

produces many copies of highly fluorescent molecules.
 Labeling in Instrumental Analysis

• Isotopic analysis
the isotopic ratio of an atom present in the analyte can be modified
using either radioisotope or a stable isotope

• Immunoenzymatic / immunochemical analysis

an enzyme or a fluorescent derivative is used as a labeling reagent

• Electrochemical analysis
redox-active reagents are frequently used.
Radioactive Decay Products
 Radioactively Labeled Molecules

molecules labeled with 14C

IEC (internal electron capture): AZX AZ-1Y*  AZ-1Y

 Radioactive Decay
Nuclear decay processes follow first-order kinetics: -dN/dt = kN

Integrating this equation we obtain: N = N0 exp(-kt)

N: number of atoms left at time t;
N0: initial number of atoms
k: characteristic decay constant

Radioactive half-life: t = - ln(1/2) / k = 0.69315/k

In practice, it is not N that is known, but rather the activity A:

A = - dN/dt = kN,

A is reported in units of “becquerel” (Bq) (1 Bq = 1 decay per

second = 1dps).
A can be directly measured by proper detector, is related to the
concentration of the radionuclide by A = kN .
A = A0 exp(-kt)
A0: initial activity
 Detecting the Radiation by Geiger Counter

The sensor tube briefly conducts electricity when a particle or a photon of

radiation temporarily makes the gas inside the tube conductive. The tube
amplifies this conduction by a cascade effect and outputs a current pulse,
which is then often displayed by a needle or lamp and/or audible clicks.
 Detecting the Radiation

Scintillator:

Scintillator transforms b-rays into a luminescence, whose intensity is

proportional to the number of b particles.

Scintillation counter
 Labeling with Stable Isotopes

A known concentration of caffeine-d3 is added to a known volume of the sample.

During GC/MS (or HPLC/MS), both forms of caffeine have same retention time. The
MS gives the intensities of the peaks at m/z 194 and m/z 197. the areas under the
respective peaks are determined. A calibration curve can be used to obtain the
concentration of caffeine.
 Immunoenzymatic Method
Antigens can induce an immune response (e.g., allergy). Introduction into the body
(human, animals, etc.) of antigen can induces the production of biomolecules called
antibodies.

Antibodies are glycoprotein (sugar-protein conjugate), also referred as

immunoglobulins. Antibodies are Y-shaped units, composed of four polypeptide chains.
Antibodies can be divided into five classes: IgG, IgM, IgA, IgD, and IgE.

The specific association of antigens and antibodies is dependent on hydrogen bonds,

hydrophobic interactions, electrostatic forces, and van der Waals forces. These are all
bonds of a weak, non-covalent nature, yet some of the associations between antigen
and antibody can be quite strong. Antigen-antibody binding is reversible.

The antibody-antigen interaction

is the basis for a variety of
important analytical procedures,
for example, ELISA.
 Enzyme Linked Immuno Sorbent Assay
secondary marker
primary antibody
antibody

target molecule

An ELISA to test for the presence of herpes

simplex virus (HSV) antibodies in blood
samples. Wells were coated with an HSV
antigen, to which antibodies against HSV
(i.e., primary antibody) will bind. The
secondary antibody is anti-human IgG
linked to horseradish peroxidase. Blood
sample with greater amounts of HSV
antibody turns brighter yellow.
 Validity of the Assay

Assay (Merriam-Webster):
1. Examination and determination as to characteristics (as weight,
measure, or quality)
2. Analysis (as of an ore or drug) to determine the presence,
absence, or quantity of one or more components; also: a test used in
this analysis
3. A substance to be assayed; also: the tabulated result of assaying

Internationally accepted assay criteria:

precision, accuracy, linearity, sensitivity, selectivity
 Signal Amplification by Enzymes
Horseradish peroxidase is often used as the “marker”, i.e., the label, in many analytical
procedures. Horseradish peroxidase is an enzyme purified from root extracts of
horseradish by affinity chromatography.

Horseradish peroxidase has a molecular weight of 44k Dalton. It appears as brown

powders, can form 10 mg/ml aqueous solution.

The oxidation of TMB is stopped by adding acid into the solution, which also
change the blue color of the product to yellow.

Luminol is oxidized in the presence of horseradish peroxidase and hydrogen

peroxide to form an excited state product 3-aminophthalate*. The 3-
aminophthalate* emits light at 425 nm as it decays to the ground state.
 Labeling Methods with Enzymes

(SMCC: N-Succinmidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate )
 Labeling with Fluorochromes

protein-NH2

protein
Fluorescein isothiocyanate (FITC) Thiourea bond formation

protein-NH2

Texas red protein

sulphonyl chloride Sulphonamide bond
formation
 The Concentration / Absorbance Relationship

N: total number of binding sites on the wall

n*: number of labeled molecules adsorbed
 Electrochemical Labeling

Fe(II)(C5H5)2  [Fe(III)(C5H5)2]+ + e

Probe:
(s = phosphorothioate linkage)
Target:
Control:

T. Ihara, et al. Chem. Commun., 1997, 1609

Hydrogen-Bonding Base Pairs in DNA
Electrochemical Labeling

The electrochemical gene

sensing system based on the
formation of complementary
sandwich-type complex

T. Ihara, et al. Chem. Commun., 1997, 1609

 Electrochemical Labeling
A significant anodic peak due to oxidation of the ferrocene moiety
was observed for the electrode treated with the target DNA.

target

control

T. Ihara, et al. Chem. Commun., 1997, 1609

 Labeling with Nanoparticles
Nanoparticles can be used as a labeling tag. For example, Fe 3O4
nanoparticles are superparamagnetic and CdSe nanoparticles emit strong
fluorescence.
The signal generated by Au nanoparticles can be: (1) colorimetric; (2)
microgravimetric; (3) electrical.

Thiols (-SH) are frequently used on

Au nanoparticles absorb light at different
wavelengths depending on their diameter
due to their size dependent surface
plasmon resonance frequency.
noble metal substrates because of the
strong affinity of sulfur for these metals.
The sulfur gold interaction is semi-
covalent, on the order of 100 kJ/mol.
 Labeling with Gold Nanoparticles

probe molecule2
probe molecule1

T. A. Taton, C. A. Mirkin, R. L. Letsinger, Science, 2000, 289,1757

Labeling with Gold Nanoparticles

After hybridizing with After exposure to Ag

Before
10 nM target amplification for 5 min
hybridizing
oligonucleotide

Before After hybridizing After exposure

hybridizing with 0.1 nM target to Ag
oligonucleotide amplification
for 5 min

T. A. Taton, C. A. Mirkin, R. L. Letsinger, Science, 2000, 289,1757