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 Velcro"

1. Simple protein immobilisation onto surfaces by multi-point avidity metal chelation
Yang L, Ling T, Vukovic P, Jennins D, Wen Ooi H, de las Heras R, Baumgartner T, Chung E, Ohse BT, Gao Y, Cooper S, Wong A, Munian C, Huang CY, Maeji, NJ
Anteo Diagnostics Ltd, Brisbane, Australia; www.anteodx.com
Introduction
Surface attachment of biomolecules onto solid supports
utilised in biosensors is increasingly challenging, due in
part to the broadening range of surfaces used (e.g.
plastics, metals, glass and ceramics) and their
continued miniaturisation.
The use of conventional approaches such as passive
adsorption and covalent chemistries can result in poor
protein stability and poor control of loading density.
Together, these can adversely damage the immobilised
protein’s structure and function limiting the sensitivity of
the biosensor.
We have developed an alternative approach that
utilises a multi-point avidity metal chelation based
surface chemistry, named Mix&Go™ (Fig. 1). Mix&Go is
comprised of cationic metal polymers (<5,000 D) that
bind surfaces with electron donating potential, resulting
in the formation of thin film coatings (approx. 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 molecular glue is 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 surfaces whilst;
1. controlling loading,
2. retaining protein functionality, and
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 incubated at room temp. for
60 min. Surface pre-treatment is not required (e.g.
COC/COP plasma treatment for passive adsorption)
and both particles (Fig 2. A) and planar surfaces (Fig 2.
B) can be activated in a similar manner. Once activated,
surfaces may be stored long term or directly coupled
with protein solution.
A.
B.
Figure 2. Mix&Go activation and protein coupling.
A. Particles may be activated and coupled in as little as 2 hr
compared to 16 hr typically required when using either Amide or
Tosyl chemistries. B. Planar surfaces such as 96-well ELISA
plates may be activated, coupled and blocked in 2.5 hr compared
to overnight coating protocols typical for passive adsorption of
proteins onto ELISA plates.
Synthetic surface
with electron donating groups e.g. OH or COOH etc
M M M
Antibodies, Proteins, Nanoparticles, Quantum dots with
electron donating groups e.g. OH, COOH etc
M
M
MM
M
Results: Controlled Protein Loading
Streptavidin (SAv) and mouse IgG were co-coupled
onto Mix&Go activated magnetic particles (Fig. 3 A &
B; 200 nm, EMD Millipore) whilst SAv and Hepatitis B
virus surface antigen (HBsAg) were co-coupled onto
Mix&Go activated plates (Fig. 3 C & D; Greiner low bind
polystyrene). Both the Mix&Go activated magnetic
particles and plates can bind two proteins
simultaneously. Importantly, the amount of coupled
protein can be controlled by a simple protein titration
step during the co-coupling process due to the
formation of a protein monolayer on the Mix&Go
activated surface.
A. B.
C. D.
Figure 3. Controlled protein loading onto Mix&Go activated
particles and plates.
A. Illustration of Mix&Go activated magnetic particles co-coupled
with a fixed SAv conc. and mouse IgG titration. B. Fluorescence
assay of titrated mouse IgG used in the co-coupling process.
Signal output (CV< 15%, amount of mouse IgG on the particle)
correlates with the mouse IgG coupling concentration. C.
Illustration of Mix&Go activated microtitre plate simultaneously
coupled with a fixed SAv conc. and HBsAg titration. D.
Colorimetric biotinylated-HRP ELISA assay and mouse anti-
human HBsAg ELISA loading assay demonstrating controlled
protein coupling onto Mix&Go.
Results: Protein Functionality
Analysis of protein activity and loading of SAv and TNF-
alpha (Fig. 4) bound onto Mix&Go activated polystyrene
surfaces (Greiner low bind) result in excellent functional
retention as compared with passive adsorption.
Synthetic substrates commonly used in biosensor
applications are available in a range of grades with
variable hydrophillic/hydrophobic profiles. We have
created a library of Mix&Go reagents matched to
specific synthetic substrates resulting in a universal
multi-point avidity metal chelating surface regardless of
the bulk properties of the underlying substrate.
A. B.
C. D.
Figure 4. Comparison of SAv and TNF-alpha protein loading and
activity using colorimetric ELISA assays.
Protein coating concentrations are indicated in each panel. A.
Direct SAv loading assay determined using biotin-HRP. B. SAv
activity assay using biotin-mouse IgG followed by detection with
goat anti-mouse-HRP. C. Direct mouse anti-human TNF-alpha
loading assay determined using goat anti-mouse-HRP. D.
Sandwich assay using human TNF-alpha (1,000 pg/mL).
0"
10"
20"
30"
40"
50"
0" 10" 20" 50" 100"
MFI"(Goat"an12mouse"RPE)"
Thousands"
Mouse"IgG"co2coupling"conc."(µg/mg"bead)"
Titra1on"of"mouse"IgG"for"co2coupling"
Batch"1"
Batch"2"
Mix&Go activated
Streptavidin
Mouse IgG
Magnetic
Particle
SAv
Results: Multi-functional Constructs
Multi-point avidity metal chelation allows generation of
novel multifunctional constructs.
Example 1: Horseradish peroxidase (HRP) and mouse
anti-human TNF-alpha simultaneously coupled to
Mix&Go-activated magnetic particles (200 nm, EMD
Millipore). The direct TNF-alpha assay result indicates
that the particle conjugate can bind TNF-alpha protein
coated on an ELISA plate (Fig 5).
A. B.
Figure 5. Generation of enzyme-antibody multifunctional
particles.
A. Illustration of Mix&Go activated magnetic particles coupled
with HRP and anti-TNF-alpha. B. Luminescence assay of titrated
TNF-alpha coated on an ELISA plate. Blank-corrected signal (CV
<10%) correlates with the TNF-alpha plate coating concentration.
Example 2: 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).
Example 3: Gold nanoparticles (10 nm, Sigma) were
coupled to Mix&Go activated magnetic particles (200
nm, EMD Millipore) and subsequently bound to mouse
IgG. The conjugate was functional as determined using
a lateral dipstick showing that the mouse IgG on the
particles are detected by goat anti-mouse whilst
remaining negative in the control (Fig. 7).
A. B.
Figure 7. Generation of a magnetic-gold conjugate.
A. Illustration of Mix&Go activated magnetic particles coupled
with gold nanoparticles and bound to a target antibody. B. Lateral
flow dip-stick analysis of antibody negative control (left) and goat
anti-mouse capturing line (indicated by an arrow).
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 can bind to both particles and planar surfaces
whilst; i) controlling loading, ii) retaining protein
functionality, and iii) creating multi-functional constructs
composed of two or more proteins/particles in a single
coupling reaction. Mix&Go is an enabling technology
that combines the ease of handling of passive
adsorption with the strength of binding of covalent
chemistry. The ease of use and versatility of Mix&Go
shows great applicability to many biosensor
applications.
Mix&Go activated
Horseradish
peroxidase
An#$TNF$α"
Magnetic
Particle
HRP
0"
2"
4"
6"
8"
10"
0.39" 6.25" 100"
RLU"(HRP)"
Thousands"
TNF=α"coa?ng"concentra?on"(ng/ml)"
Magne?c"Ab=HRP"conjugate""
Batch"1"
Batch"2"
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
Qdot linked
Mix&Go 200 nm
magnetic particles
200 nm
magnetic particles
5570
1
Mix&Go activated
Gold nanoparticle
coupled
Target
Molecule
Magnetic
Particle
Au
10nm