Scientific poster presented at the 10th International Conference on the Scientific and Clinical Applications of Magnetic Carriers (MagMeet) in Dresden by Anteo Diagnostics. Data is provided that shows that Mix&Go activated particles can bind to various biomolecules simultaneously such as antibodies (e.g. IgG, IgM, Fab, single domain Ab), proteins (e.g. streptavidin, Protein A/G, enzymes, BSA), nucleic acids (e.g. ssDNA) or other nanoparticles (e.g. gold nanoparticles, quantum dots)

1. Simple Water Based Protein Conjugation and/or Assembly of Complex
Magnetic Nanoparticles
C.Y. Huang, E. Chung, S.J. Cooper, R. de las Heras, B.T. Ohse, Y. Gao, D. Jennins, T. Ling, C. Munian, L. Yang, P. Vukovic, A. Wong, N.J. Maeji
Anteo Diagnostics, Brisbane, Australia; www.anteodx.com
Introduction
Conventional approaches such as passive adsorption
and covalent chemistries to attach biomolecules to
nano and micro particles can result in poor control of
protein loading density, its functional stability and
reproducibility. Another complication is the tendency of
particles to clump or aggregate during the process
especially with smaller particles.
We have developed a simple, water based alternative
approach based on metal chelation that resolves such
difficulties. Affinity of metal complexes to a ligand vary
enormously from less than femtoseconds to many
hours. Polymerisation of the latter type of metal
complexes to form oligomers enable avidity or multi-
component binding of proteins to almost any particle.
Named Mix&Go™ (Fig. 1), these cationic metal
polymers (<5,000 D) bind surfaces with any electron
donating potential, resulting in the formation of thin film
coatings (~1 nm thickness) that are very stable and
robust. The polymeric metal ions of Mix&Go, chelate
and bind by multi-avidity to both the synthetic surface
and to biomolecules, thereby acting as a ”molecular
glue".
Figure 1. Mix&Go, a molecular glue comprised of
polymeric metal ions that chelate to available electron
donating groups on synthetic surfaces and proteins.
We demonstrate the utility of using Mix&Go to couple
proteins onto several different particles whilst:
1. Controlling loading
2. Retaining protein functionality
3. Creating multi-functional constructs composed of
two or more proteins/particles in a single reaction
Methods
Mix&Go is an aqueous solution that is directly applied
to synthetic surfaces and normally incubated at room
temperature for 60 minutes. However, Mix&Go binding
to Life M-270 Dynabeads has been shown to be
complete within 1 minute. In order to prevent any cross-
linking, the particles are slowly added to an excess of
Mix&Go. On removal of any unbound Mix&Go, the
positively charged particles maintain their colloidal
stability via charge-charge repulsion.
Once activated, the particles may be stored long term,
as they are indefinitely stable. However, in the
presence of proteins, the pre-existing water molecules
are rapidly displaced to form a very strong and gentle
interaction between the protein and the particle.
Mix&Go can bind to any available electron donating
groups on the particle so any particle including silica or
any metal oxides, can be directly activated to bind
proteins.
Figure 2. Mix&Go activation and protein coupling.
Particles may be activated and coupled in well under 2
hours.
Results: IgG and SAV Co-Coupling
Rapid binding kinetics of proteins to Mix&Go activated
particles allow simple control over protein loading by
amount of protein added. For example, Streptavidin
and mouse IgG were co-coupled to Mix&Go activated
magnetic particles (200 nm, EMD Millipore)
simultaneously. The results in Fig. 3 show that Mix&Go
activated magnetic particles can bind to streptavidin
and mouse IgG simultaneously. The coupling amount of
mouse IgG is controlled by a simple IgG titration step
during the co-coupling process. The lateral flow dipstick
result shows that the antibodies on the particles are
detected by goat anti-mouse and stay negative in the
control.
A.
C.
Figure 3. A. Illustration of Mix&Go activated magnetic
particles coupled with streptavidin and mouse IgG. B.
Fluorescence assay of titrated mouse IgG used in co-
coupling process. Signal out-put (%CV< 15%, amount
of mouse IgG on the particle) correlates with the mouse
IgG coupling concentration. C. Lateral flow dip-stick
analysis of antibody negative control (left) and the goat
anti-mouse capturing line (indicated by an arrow).
Results: IgG and HRP Co-Coupling
Horseradish peroxidase (HRP) and mouse IgG are co-
coupled to Mix&Go-activated magnetic particles (200
nm, EMD Millipore) simultaneously. The results in Fig.
4 show that Mix&Go activated magnetic particles can
bind to HRP and mouse IgG simultaneously. The direct
TNF-a assay result shows that the anti-TNF-a antibody
on the particles can detect TNF-a protein coated on an
ELISA plate. This demonstrates that HRP and anti-
TNF-a antibody have been co-coupled to one particle
simultaneously.
A. B.
Figure 4. A. Illustration of Mix&Go activated magnetic
particles coupled with streptavidin and mouse IgG. B.
Fluorescence assay of titrated TNF-a coated on an
ELISA plate surface. The blank-corrected signal output
(%CV <10%) correlates to the TNF-a coating
concentration on the plate.
0
10
20
30
40
50
0 10 20 50 100
MFI(Goatan-mouseRPE)
Thousands
Mouse IgG co-coupling conc. (mg/mg bead)
Titra on of mouse IgG for co-coupling
Batch 1
Batch 2
Mix&Go activated
Streptavidin
Mouse IgG
Magnetic
Particle
SAv
Results: IgG on Gold Nanoparticles
Gold nanoparticles (10 nm, Sigma) were coupled to
Mix&Go activated magnetic particles (200 nm, EMD
Millipore) and can subsequently bind to mouse IgG
successfully. The results from Fig. 5 show that Mix&Go
activated magnetic particles can bind to gold
nanoparticles. The lateral dipstick result shows that the
mouse IgG on the particles are detected by goat anti-
mouse and stays negative in the control.
A.
C.
Figure 5. A. Illustration of Mix&Go activated magnetic
particles coupled with gold nanoparticle and binding to
a target antibody. B. Comparison of UV/VIS scanning of
before and after gold nanoparticle coupling to Mix&Go
activated magnetic particles. C. Lateral flow dip-stick
analysis of antibody negative control (left) and goat
anti-mouse capturing lines (indicated by an arrow)
Results: Quantum Dot Coupling
Mix&Go multi-point avidity metal chelation can also be
used to couple two synthetic substrates together as
illustrated using Quantum dots bound onto magnetic
particles (Fig. 6).
A. B.
Figure 6. Generation of Qdot-magnetic particles.
A. TEM image of a bare magnetic particle and a
quantum dot-coupled magnetic particle (indicated by
arrows). B. Illustration of Qdot-linked magnetic particles
showing signal versus background (5570 : 1).
Conclusion
Mix&Go is a simple universal coupling technology that
utilises multi-point avidity metal chelation to bind
surfaces with electron donating potential thereby acting
as a “molecular glue”.
Mix&Go activated particles have been proved to bind
various biomolecules simultaneously such as
antibodies (e.g. IgG, IgM, Fab, single domain Ab),
proteins (e.g. streptavidin, Protein A/G, enzymes, BSA),
nucleic acids (e.g. ssDNA) or other nanoparticles (e.g.
gold nanoparticles, quantum dots).
A novel multi-purpose binding platform facilitated by
Mix&Go comprising pre-activated nanoparticles can
provide a very easy to use and powerful tool kit for
researchers to generate multi-functional nanoparticles
effectively and efficiently to explore myriad possibilities.
Mix&Go activated
Horseradish
peroxidase
An -TNF-a
Magnetic
Particle
HRP
0
2
4
6
8
10
0.39 6.25 100
RLU(HRP)
Thousands
TNF-a coa ng concentra on (ng/ml)
Magne c Ab-HRP conjugate
Batch 1
Batch 2
Synthetic surface
with electron donating groups
such as OH or COOH etc
M M M
Antibodies, Proteins, Nanoparticles,
Quantum dots with electron donating
groups such as OH, COOH etc
M
M
MM
M
Negative
control
Goat
anti-
mouse
IgG
Test line
Mix&Go activated
Gold nanoparticle
coupled
Target
Molecule
Magnetic
Particle
Au
10nm
0
1000
2000
3000
4000
5000
6000
200nm Mix&Go
activated
magnetic beads
Qdot linked
Mix&Go
magnetic beads
MFI
Qdot 800 (Life Technologies) linked
Mix&Go beads
Background vs Signal
B.
B.