Nano Biotechnology

Enabled Point of Care

Anil Kumar - 19BT10019
Akhilesh Kumar - 18CY20003
Poorvi Asthana - 22MM62R12
Group 2 S.B Tanay Gaurav - 19CH10038
Gourav Chauhan - 22MM44002
Anjana Devi D S - 22BT60R12
Gopika K M - 22EE62R04
High sensitive gold-nanoparticle based lateral flow
Immunodevice for Cd2+ detection in drinking waters
Contents
➔ Introduction
➔ Abstract
➔ Working Principle
➔ Preparation of AuNP for LFID
➔ Results
➔ Conclusions
Introduction

● Heavy metals are not biodegradable. They have been isolated from soil, drinking
water and surface and ground waters in residential areas.
● Especially Cd2+ is one of the most harmful heavy metal. It can be accumulated in
the human body mainly in the liver and kidneys.Depending upon the ingestion
route, Cd2+ exposure can affect kidney, lung function, or produce cardiovascular
diseases.
● low-level chronic Cd2+ exposure, in combination with other environmental factors,
can contribute to an increased risk of cancer.
Contd
● As it is well-known graphite furnace atomic absorption spectroscopy (GFAAES)
and inductively coupled plasma emission spectroscopy (ICPES) are the standard
techniques for trace heavy metal analysis.
● Here a novel and high sensitivity lateral flow immunoassay device (LFID) for
Cd2+ detection in aqueous samples using the Cd–EDTA–BSA conjugate labeled
with gold nanoparticles as signal producer tool is implemented.
● The developed LFID is based on the recognition of Cd–EDTA complex (formed
after Cd2+ analyte complexation) by using the specific 2A81G5 monoclonal
antibody (mAb).
● The EDTA molecule has six coordinative sites. It is a hexadentate ligands
● It is used in medicine to prevent blood samples from clotting and to remove calcium
and lead from the body.
● It is a type of chelating agent. Also called edetic acid and
etheylenediaminetetraacetic acid.
Abstract
● In this work for first time a lateral flow immunosensor device (LFID) for Cd2+
determination in drinking and tap waters using the Cd–EDTA–BSA–AuNP
conjugate as signal producer tool is introduced.

● The principle of working is based on competitive reaction between the

Cd–EDTA–BSA–AuNP (Bovine serum albumin (BSA ))conjugate and the
Cd–EDTA complex.

● the Cd–EDTA–BSA–AuNP conjugate deposited on the conjugation pad strip and

the Cd–EDTA complex formed in the analysis sample for the same binding sites of
the 2A81G5 monoclonal antibody, specific to Cd–EDTA but not Cd2+ free, which
is immobilized onto the test line.
● The device has a large response range within 0.4–2000 ppb, being the
linear response between 0.4 and 10 ppb.

● The quantification and detection limits of 0.4 and 0.1 ppb,

Respectively.

● The obtained detection limit is 50 times lower than the maximum

contamination level required for drinking water.

● Here we also show a new option for increasing the sensibility in the
LFDs with competitive format.
● We achieve through the decreasing concentrations of the
Cd–EDTA–BSA–AuNP conjugate deposited in the conjugation
strip and the mAbs deposited in the test and control zones until to
reach optimized concentrations.

● It is an important result take into account that the increase in sensibility is

one of the challenges in the field of LFD sensors.
Working principle of AuNP based LFID for
Cd2+ detection
● The principle involved in the working of AuNP based LFID for Cd2+ detection is
the “Competitive Binding or Affinity Binding.”
● Competitive binding is a type of preferential binding phenomenon among two
molecules or analytes for binding to a same target molecule.
● The two analytes compete with each other for binding to the same target molecule
for the same binding sites.
● The molecule to be analysed is the ‘analyte’ and the molecule that is targeted to
bind is the ‘target’.
● Phenomenon of competitive binding occurs in many cases like Enzyme kinetics
(competitive inhibition), various immunological assays.
● Preferential binding by the target molecule to a particular analyte is based on the
higher binding affinity it has for that particular analyte as compared to the analogous
molecule.
● The extent of binding relationship between a biomolecule (ex. Protein or DNA) and
its ligand/binding partner is referred to as the binding affinity.
● KD (binding/dissociation constant) is the constant that is used to measure and
report the binding affinity.
● The lower the KD value, the higher the analyte’s binding affinity to the target
molecule.
● It can be also said as the change in free energy associated with a binding process.
● It is a type of Equilibrium constant K, and is the inverse of the KA (association
constant).
● Lower the value of Kd, higher the affinity of the ligand with the receptor.
● The binding takes place in three subsequent phases, i.e., association , equilibrium
and dissociation
● Factors responsible for the binding affinity of the ligand & receptor are:
- Electrostatic interactions, non-covalent interactions like hydrogen bonding, Van der
waals forces etc.
- It can be measured as the rate constant at which the ligand dissociates from the
receptor
- Its unit is mol/lit.
- Cognate functional groups like NH2 , COOH group etc.
- pH, temperature, surface area etc of the reaction system.
● In context of our AuNP based Cd2+ detection LFID, the competitive binding takes
place between Cd-EDTA complex and Cd-EDTA-BSA-AuNP conjugate, for binding
to the same monoclonal antibody .
 Preparation of Au-NPs for the LFID
Approx. 20 nm dia AuNPs dissolution synthesized according to the citrate reduction of
HAuCl4 dissolution.
Contd.
● HAuCl4 (aq. soln) heated to boiling and vigorously stirred.
● Sodium citrate added to it & boiling continued for some time.
● Solution cooled to room temp. with a continuous stirring
● Colloids stored in dark bottles at 4 deg C. Why?
 Preparation of Cd–EDTA–BSA–Au conjugate
● The pH of AuNPs-citrate dissolution was adjusted to 7.4 with NaOH.

In a typical labeling of the Cd–EDTA–BSA conjugate with AuNPs, AuNPs-citrate

pH 7.4 was incubated with Cd–EDTA–BSA conjugate in HEPES buffered saline
(HBS: 137mM NaCl, 3mM KCl ,and 10 mM HEPES, pH 7.4).
Why buffering???

Wash-free biosensor!
 Preparation of LFID
● A conjugation strip of length 18 cm cut.
● Soaked in conjugation pad solution.
● Dried at vacuum during and stored at 4 deg C.

Conjugation pad soln: AuNP-citrate (pH7.4) incubated with Cd–EDTA–BSA conjugate

( pH 7.4).

● Process: Centrifugation+Suspension
● Strip mounted on a plastic backing card for easier handling.
Strip overview
● Sample pad: Initial sample
flow.
● Conjugate pad: Holds the
detector particles stable for
test.
● Test line: Biomarker
specific Ab. Crucial for
visual detection.
● Control line: Capture
reagent with test line.
Captures all Abs. regardless
of An. being present.
● Absorbent pad: Soaks
excess sample
 Optimization of LFID overview
Optimization of the antibodies onto the test and control lines and the conjugate onto the
conjugation strip for better Cd2+ sensitivity.
3 stage optimization:
1. First optimizations of the LFID carried out by drop coating of different mAbs
concentrations onto the membrane for the same conjugation strip preparation.

2. Decreasing the concentration of the conjugate onto the conjugation strip and keeping
the same immobilized amount of the mAbs previously determined.

3. Finally the last optimizations assays were made using the automatic dispensing.
Working Mechanism

•The detection mechanism of this developed lateral flow immunosensor

device (LFID) is based on the use of a purified 2A81G5 mAb that recognizes
with high specificity Cd–EDTA complexes but not the free Cd2+ ions.

• The affinity of 2A81G5 mAb for the Cd–EDTA complex is associated with
the histidine-H96 (His-96) and tryptophan (Trp-H52) residues in the heavy
chain variable regions of this mAb being these important as antigen recognition
sites.
3-D structure of mAb

•There is closest distance of 2.72 Å between

Cd2+ and N of His-96, which suggests a
possible interaction between
antigen-antibody.

• This model also suggest hydrogen bonding

interaction b/w the nitrogen of the antigen
and the Trp-H52 residue.
Pre-run strip
 Without Cd- EDTA

● Sample flow is due to the capillary action of the strip.

● Color intensity on the test line is maximum.
 With Cd-EDTA

● Decrease in the color intensity on the test line due to decrease in the
quantity of color conjugate bound on the primary antibody.
Discoloration with low analyte concentration

● Higher quantity of the conjugate binds on the

large quantity of 2A81G5 mAb and does not
allow the notable discoloration.

● By reducing the concentrations of the

conjugate onto the conjugation pad and the
2A81G5 mAb onto the test line we can
obtain a notable discoloration at low analyte
concentration.
Results : A) Gold aggregation test for the
Cd–EDTA–BSA conjugate

(1) AuNPs+Cd–EDTA–BSA,
(2) AuNPs+Cd-EDTA-BSA+1.2% NaCl,
(3) AuNPs+Cd–EDTA–BSA+5.5% NaCl.
 (B) Absorption spectra of Cd–EDTA–BSA–AuNPs
conjugates

(1) 135 µL AuNPs+15 µL HBS,

(2) 135 µL AuNPs+15 µL
Cd–EDTA–BSA (80 mg/mL)
(3) 135 µL HBS+15 µL
Cd-EDTA-BSA (80 mg/mL).
( C) TEM Images of AuNPs

(1) AuNP (2) Cd–EDTA–BSA–AuNP

Typical image of the Cd2+ detection by the LFID
(B) Lineal range of the device for Cd–EDTA analysis.
(C) Sensitivity over the entire range of the device for
Cd–EDTA analysis.
Analysis of Interfering effect with and
without OVA

(A) Interference of the Ca2+, Mg2+, Fe3+ (B) Interference of the Co2+, Ni2+, Zn2+ and
and Al3+ ions in presence of 10 ppb Cd2+ Cu2+ ions in presence of 10 ppb Cd2+
Analysis of Interfering effect with and
without OVA

(C) Interference of the Mn2+ and

(D Effect of the interference produced by 10%
Hg2+ ions in presence of 10 ppb
OVA when 100 and 10 ppb Cd2+ are analyzed.
Cd2+
CONCLUSION

● Highly sensitive lateral flow device for Cd2+ detection in drinking water
samples.
● Based on the principle of competitive binding.
● Cd–EDTA–BSA conjugate labeled with Au NP is responsible for the
signal/indication produced in the test line.
● The device was also studied for several metal interferences.
● Optimizing the mAb and conjugate concentration as well as strip length
leads to large operating range and low LOD and LOQ.
ADVANTAGES:
● Wash-free biosensor.
● Large response range (0.4 - 2000 ppb).
● Notably low Limit of Detection and Limit of Quantification.

Detection limit is 50 times lower than the minimum allowed level of Cd in

drinking water.

● Increased sensitivity owing to its competitive format.

● Low or manageable interference from other metal ions.
● Simple, low cost and Point-of-Care method, unlike other conventional
methods of Cd detection.
FUTURE PROSPECTS:
● Integration of interference removal process within the LFD.
● Working principle of the device may be extended to applications in
diagnostics and safety.
● NP size and surface chemistry can be optimized further to improve
sensitivity and specificity.
● Integration with digital technologies: There is growing interest in
connecting lateral flow immunoassay devices to digital platforms, such as
smartphones or cloud-based systems, to enable remote monitoring and
data analysis.
References : High sensitive gold-nanoparticle based lateral
flow Immunodevice for Cd2+ detection in drinking waters

Authors : Adaris M. López_Marzo, Josefina Pons, Diane A. Blake, Arben Merkoc-i

Journal : Biosensors and Bioelectronic