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

M M 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 part ic les 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"(G

oat"a

n12m

ouse"RPE

)"Th

ousand

s"

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 applicabil i ty 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)"

Thou

sand

s"

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