Analytical Sensing Technologies Immunoassays

1. Immunoassays
An analytical technique that uses antibodies as reagents to quantify
specific analytes $6 billion industry worldwide
The sample can be
Clinical urine, blood, saliva 2.5 billion tests sold annually
Environmental / Agricultural water, soil extracts, plant extracts,
animal products/tissues (blood, urine, milk, meat), food, industrial Highly quantitative
processes and effluents
Regulatory approved
2. DNA sensing technology
A group of techniques for detecting DNA hybridisation
Drug discovery
Flexible test formats
Pathogen detection
Forensic identifications Highly reliable

Immunoassays Immunoassays
Diverse markets and applications References
Clinical diagnostic immunoassays Recent Developments in Electrochemical Immunoassays and
! In use > 30 years Immunosensors, J.M.Fowler, D.K.Y.Wong, H.B.Halsall,
! Basis for critical human health decisions W.R.Heineman, in Electrochemical Sensors, Biosensors, and Their
" Disease diagnosis (AIDS, hepatitis, prostate-specific Biomedical Applications, Edited by X.Zhang, H.Ju and J.Wang,
antigen (PSA)) Chapter 5, page 115-143, Elsevier, 2008. [R857.E52.E54]
" Therapeutic drug monitoring
Strategic Applications of Nanomaterials as Sensing Platforms and
" Drugs of abuse screening
Signal Amplification Markers at Electorchemical Immunosensors,
" Over 70 clinical analytes tested by immunoassays
D.K.Y.Wong and others, Electroanalysis, DOI: 10.1002/elan.
" Home pregnancy tests
201600166.
Agricultural
Environmental Analytical Chemistry, Edited by R.Kellner, J.-M.Mermet, M.Otto,
Food H.M.Widmer Wiley-VCH (1998) [QD75.2.A63], page 405-429.
Industrial
Pharmaceutical
Veterinary
Water quality
Antibodies Antibodies

Key reagents in all immunoassays A major class of large proteins known as immunoglobulins (Ig),
Proteins produced by immune system of higher animals usually consisting of four peptides arranged in two pairs two
o Produced by specific white blood cells specific combining sites capable of interacting with an antigen
o In response to recognition of foreign substances IgG is the most abundant antibody (~70%) and is of a Y-
o Examples: vaccinations; response to natural infections shaped structure: epitode Antigenic
(mumps, chicken pox) binding
site

NH 2

NH 2
NH 2

NH 2
Physically bind to antigens Disulfide
bridges
Tightly bind only to substance that elicited production Light (L)
chain
Strength of binding (affinity) determines sensitivity of SS
SS S S

method HOOC SS COOH

Specificity allows detection in complex matrix Hinge region

o Minimum sample preparation Heavy (H) chain

HOOC COOH

Interaction between an Antibody (Ab) and an Interaction between an Antibody (Ab) and an
Antigen (Ag) Antigen (Ag)

Primary binding force is ionic (long range interaction) over Note that [Ab]total = [Ab] + [Ab-Ag]
~10 nm " !"! !#$
# %
Following slow exclusion of hydration water, hydrogen = $ %& "# !"$%
" !#$
# %
bonding forms
= $ %& "# !"$% !$ %& "# !"! !#$%
Van der Waals forces (short range interaction) operating '(')*

between 0.5-0.15 nm, strengthening the bonds

Use this equation to construct
" #$! #%$ a Scatchard plot.
Consider Ab + Ag Ab-Ag K !" =
# %
" #$$" #%$
# %# %
Typical values of Keq: 106 1012 (limited use in immunoassay if
<108).
 Interaction between an Antibody (Ab) and an Interaction between an Antibody (Ab) and an
Antigen (Ag) Antigen (Ag)

Monoclonal versus Polyclonal Purpose of immunoassay is to follow the equilibrium between

Monoclonal Polyclonal
Ab and Ag.
Lot-to-lot consistency Lot-to-lot variability
Investigate the binding between Ag and Ab and distinguish
Indefinite supply More broadly reactive
between bound and unbound Ag.
Highly specific Often more sensitive
Longer lead time Shorter lead time As this is an equilibrium reaction, it is not linear with Ag
Higher initial costs Lower initial costs concentration.

It is important to know the characteristic binding response, as a

Selection is based on application, time and cost
function of [Ag].

Interaction between an Antibody (Ab) and an

Antigen (Ag) Immonucomplexes for detecting binding
" #$! #%$
From K = # % and [Ag]
total = [Ag] + [Ab-Ag]
!" " #$$" #%$
# %# % Detectable label
" #$! #%$
# %
K !" =
{
" #$$ " #%$ ! " #$! #%$
# % # %&'&() # % } Detectable label
& * !" "# #$$%"# #%$% !* !" "# #$$%"# #$! #%$% = "# #$! #%$%
Radiolabel
&'&()
Immunocomplex Enzyme
& * !" "# #$$%"# #%$% =+,+ "# #$$%* !" -"# #$! #%$% Fluorescence
&'&()
" #$! #%$ " #$$* Luminescence
# % # % !" ,
&
" #%$
=
" #$$*
= Electrochemical
# % ,+ "
# % !" ,+, # #$%* !"
$
{ }
&'&()

Then
! Ab - Ag# ! Ab#K ! Ab - Ag# ! Ag# antibody
" $ 1 1 " $ eq " $ " $
= = = =
! Ag#
" $ 1+
1 ! Ab#K +1
" $ eq
! Ab#K +1
" $ eq
! Ab - Ag#
" $ antigen
total
! Ab#K +1
" $ eq ! Ab#K ! Ag#
" $ eq " $

Immunoassay formats
1. Competitive Immunoassay
I. No analyte high detection signal
II. Analyte present detection signal reduced
Immunoassay formats
2. Non-competitive (sandwich) immunoassay
Interaction between an Antibody (Ab) and an
Antigen (Ag)
It gives a measure of sites not
occupied by analyte
The analyte is mixed with labelled
analyte and they compete for
binding sites
This assay requires separation of
bound and free antigen or antibody,
in order to determine their relative
amounts
For an antigen analyte, the presence
of labelled antigen provides a means
of assessing the relative partitioning
Optimum sensitivity can be obtained
by decreasing the concentration of
labelled antigen and antigboy
Immunoassay formats
The analyte antigen is captured by a
capture antibody (Ab1), allowing it to be
separated from the rest of the sample
The Ab1-Ag complex is then incubated with
excess of a second, labelled antibody
(signal antibody), Ab2, which binds only to
the existing Ab1-Ag complex
Ab2 must be able to bind specifically to an
exposed surface of the bound antigen
Ideally, there would be no signal in the
absence of antigen, because there would
be no binding site available for the labelled
antibody
In practice, there is always some signal
arising from non-specific binding of Ab2 to
Ab1, even in the absence of total signal
Common Labels used in Immunoassays Common Labels used in Immunoassays

How to attach these labels?

Radioactive labels Using simple isotope replacement
125I a !emitter and 3H (tritium) a -emitter
Using radiommunoassay (RIA) based on Chloramine-T or the
enzyme, lactoperoxidase (LPO) for peptides and proteins
However, 3H is less sensitive than 125I because particles emitted are
weak and are largely absorbed by the walls of the vessel. that have an aromatic ring
CH3 CH3
Na125I
! rays are not absorbed by the vessel, and the counting efficiency, using Chloramine-T; pH 7.5
a scintillation counter, is typically of the order 80%. OH OH
125I

Incorporation of iodine with Chloramine-T as oxidant

CH3 CH3
Na125I
LPO / H2O2; pH 7.5
125I
OH OH
Incorporation of iodine with lactoperoxidase | hydrogen peroxide as oxidant

Common Labels used in Immunoassays Common Labels used in Immunoassays

For peptides and haptens without an aromatic ring, prepare Using commercially available compounds (e.g. N-hydroxy-
a conjugate with a labelled molecule (e.g. tyrosine-like succinimide ester of 3-(p-hydroxyphenyl) propionic acid) to
residue) and then attach it to them. facilitate direct reaction of this reagent with free amino
OH
OH
H2 N COOCH3
OH groups to yield an amide bond coupling
CH3 CH3 CH3
O
O
O H2 N
H2NOCH2COOH I CH2 CH2 C O NH
CH I CH2 CH2 C O N
HO HO
HO
O NOCH2 CONH O HO
NOCH2 COOH HO
COOCH3
125I-

LPO | H2O2 Conjugation labelling in which N-succinimidyle 3-(4-hydroxy 5-{125I} iodophenyl)

propionate reacts with free amino groups.
OH
Labelled conjugate

Synthesis of a tyrosine methyl ester conjugate: the C6-keto group of 6

keto-17 -estradiol
Hapten
any large or small molecule that does not elicit an
immune response by itself but can be shown to
interact with an antibody
Common Labels used in Immunoassays
In general, the reagents
selected for the coupling
depend on the availability of
reactive groups in the peptide
or hapten
Typical coupling reagents for
conjugation to proteins
Common Labels used in Immunoassays
Enzyme labels
Most widely used in immunoassays
Disadvantage ! not producing direct measurement because
measurement may involve detection of the consumption of
the enzyme substrate, or the accumulation of product
Advantage ! enhanced signal because a single enzyme
molecule can cause the conversion of many substrate
molecules
Two most commonly used are alkaline phosphatase (ALP)
and horseradish peroxidase (HRP)
Similar methods for conjugation of enzyme to antibody or
antigen to those available for radiolabels but care must be
taken such that the active sites of the enzyme are not
masked!

Common Labels used in Immunoassays Signal Generation

In some cases, a spacer molecule is positioned between the The enzyme substrate should be converted to a coloured
hapten and protein to prepare an antibody-enzyme product / fluorescent product / electroactive product for the
conjugate; or to enhance binding reactivity with the respective mode of detection
antibody. Example: using glutaraldehyde as the Consider the enzyme alkaline phosphatase:
homobifunctional agent leads to heterogeneous complexes, Using p-nitrophenyl phosphate or a similar aromatic phosphate
with significant enzyme-enzyme cross-linking, as well as the as substrate:
desired enzyme-conjugatel heterobifunctional agents (N-
O- OH
hydroxy succinimide esters of dimalemide and carbodiimides) O- H2O O-
+ - +
have been shown to avoid problems N
.. P O
Alkaline phosphatase
N
.- +O P
-
OH
O O
O
p-notrphenyloxide O
(!max 440 nm)

EDC-NHS coupling reaction Avidin-Biotin coupling

Signal Generation Signal Generation

If using 4-methylumbellieryl phosphate as substrate, we can Consider the enzyme horseradish peroxidase (HRP).
produce 4-methylumbelliderone. This product emits at 448 nm Using luminol as cosubstrate with HRP produce 3-amino
when it is excited at 365 nm ! producing fluorophore; can rely phthalate that emits light can rely on chemiluminescence
on fluorescence as detection as detection
To facilitate electrochemical detection: In electrochemical detection:
HO O OH O
ALP
H2N O O +H2O H2N OH + P
P HO OH HRP
HO OH
+ H2O2 + 2H2O

p-aminophenyl phosphate p-aminophenol phosphoric acid OH O

Hydroquinone Benzoquinone
O OH

- reduction
+ 2 H+ + 2e

O OH

Signal Generation DNA biosensors

Label-free electrochemical detection Watson-Crick
X.Liu, P.A.Duckworth, D.K.Y.Wong, Biosensors and Bioelectronics, 25 DNA included four bases adenine (A), thymine (T), guanine
(2010) 1467-1473 (G) and cytosine (C).
# Rely on the ease of oxidation or reduction of a redox marker
# Cyclic voltammetry of [Fe(CN)6]3-/4- at a bare glassy carbon
electrode measure peak current (Ip1)
# Cyclic voltammetry of [Fe(CN)6]3-/4- at an antibody-antigen
modified bare glassy carbon electrode measure peak
current (Ip2) A T G C
# Expect Ip2 < Ip1 because the antibody-antigen layer prohibits
the direct contact of [Fe(CN)6]3-/4- on the glassy carbon Matching G --- C and T --- A
electrode
# More antibody-antigen complex thicker layer on electrode
the smaller the peak current quantitatively related to
the analyte antigen
 How to immobilise biomolecules on an
Design of DNA Biosensors electrode?
On a gold electrode

RESEARCH ARTICLE

www.acsami.org

Modular Click Chemistry for Electrochemically and

How to immobilise biomolecules on an
Photoelectrochemically HowInterfaces
Active Molecular to immobilise biomolecules
to on an
electrode? electrode?
Tin Oxide Surfaces
On a glassy carbon electrode Click chemistry selectively labelling or linking biomolecules
Michelle C. Benson, Rose E. Ruther, James B. Gerken, Matthew L. Rigsby, Lee M. Bishop, Yizheng Tan,
Shannon
Reduction of aryland
S. Stahl, diazonium
Robert cations
J. Hamers*
Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue Madison, Wisconsin 53706, United States

bS Supporting Information
Oxidation of 2 amines
Benson et al. ACS Applied Materials & Interfaces, 3 (2011) 3110-3119.
ABSTRACT: We demonstrate the use of click chemistry to form electrochemically and
photoelectrochemically active molecular interfaces to SnO2 nanoparticle thin lms. By using
photochemical grafting to link a short-chain alcohol to the surface followed by conversion to a
surface azide group, we enable use of the Cu(I)-catalyzed azide!alkyne [3 + 2] cycloaddition
(CuAAC) reaction, a form of click chemistry, on metal oxide surfaces. Results are shown
with three model compounds to test the surface chemistry and subsequent ability to achieve
electrochemical and photoelectrochemical charge transfer. Surface-tethered ferrocene groups
exhibit good electron-transfer characteristics with thermal rates estimated at >1000 s!1.
Time-resolved surface photovoltage measurements using a ruthenium terpyridyl coordination compound demonstrate photoelec-
tron charge transfer on time scales of nanoseconds or less, limited by the laser pulse width. The results demonstrate that the CuAAC
click reaction can be used to form electrochemically and photoelectrochemically active molecular interfaces to SnO2 and other
metal oxide semiconductors.