linkers, tethers - sensopath · summary linkers and tethers surface plasmon resonance (spr), in...
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LINKERS, TETHERS
Creating Bioactive Surfaces Effective Biomolecule Presentation Tether the biomolecule to the sensor surface
PRODUCTS AVAILABLE
ü Monothiol alkane PEG alcohols, carboxylates, hydrazides ü Biotinylated alkane thiols ü Dithiol aromatic PEG alcohols, carboxylates, hydrazides
ü NHS-terminated PEGylated benzaldehyde linkers for AFM
PEGylated silanes R&D for microfluidics, microarrays and AFM
MonothiolalkanePEG Mixed Self-assembled Monolayer Surface
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Lahiri, et al 1999 A Strategy for the generation of Surfaces Presenting Ligands for Studies of Binding Based on an Active Ester as a Common Reactive Intermdiate: A Surface Plasmon Resonance Study Anal Chem 71,777
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-CONHNH2
Self-assembled monolayers improve protein distribution on holey carbon cryo-EM supports Joel R. Meyerson1, Prashant Rao1, Janesh Kumar2, Sagar Chittori2, Soojay Banerjee1, Jason Pierson3, Mark L. Mayer2 & Sriram Subramaniam1
Poor partitioning of macromolecules into the holes of holey carbon support grids frequently limits structural determination by single particle cryo-electron microscopy (cryo-EM). Here, we present a method to deposit, on gold-coated carbon grids, a self-assembled monolayer whose surface properties can be controlled by chemical modification. We demonstrate the utility of this approach to drive partitioning of ionotropic glutamate receptors into the holes, thereby enabling 3D structural analysis using cryo-EM methods.
Modification using SPT-0011P6
Scientific Reports 18 Nov 2014
Self-assembled Monolayers Disadvantage of monothiol SAMs Monothiol surfaces tend to be unstable. Why? Monothiol SAMs dissociate from gold resulting in loss of coverage and loss of capture agent especially in a flow cell Solution: Use a dithiol SAM for excellent stability. Why? They don’t float away. Multiple points of attachment require a simultaneous event in order to completely dissociate so adsorption to gold is exponentially better.
Dithiolalkanearomatic PEG Mixed Self-assembled Monolayer Surface
Spangler, BD, and Spangler, CW 2006 Biosensors Utilizing Dendrimer-immobilized Ligands and Their Use Thereof US Patent #7,138,121 B2
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OCOOH COOH -CONHNH2
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Energy minimized geometries and molecular dimensions
Fragoso, A., Laboria, N., Latta, D., and O’Sullivan, C. K. (2008) Anal. Chem. 80, 2556-2563
EDC/NHS Chemistry
• Advantages – Simple, well worked out chemistry – Fairly rapid reactions
• Disadvantages – Blocks lysine residues anywhere in protein – Non-specific orientation
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S (CH2)15-CNH
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COUPLING TO HETEROBIFUNCTIONAL TETHER
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC) and (N-Hydroxy Succinimide (NHS) G. T. Hermanson Bioconjugate Techniques Elsevier AP 2nd Edn 2008
Aldehyde to Hydrazide-terminated Tether
• Advantages: – Unique functionality – Unique placement on glycosylated proteins – Retains antibody activity – Preferentially orients antibody – Unobstructed variable domains
• Disadvantage: Not all antibodies or proteins are glycosylated
ORIENTED COUPLING TO HETEROBIFUNCTIONAL TETHER
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NHN=
CH
SPT-0014B
S S Gold Surface
OHC
QCM Biosensor
• Surface was prepared with a 10:1 ratio dithiol alkanearomatic PEG3:dithiolalkanearomatic PEG6-COOH
• Anti-PSA and Mouse IgG Immobilization (control surface) Sierra Sensors QCMA1. • Concentrations of PSA were injected over both surfaces simultaneously. The sensor
surface was regenerated back to the immobilized protein with a 1 m injection of 100 mM HCL.
Multiplex SPR: Selectivity
Monothiol Mixed SAM-coupled antibody
A method for label-free detection of proteins in crude cell lysate by means of an antibody array and SPR imaging. The cell lysate was applied to the
array surface without dilution in order to maintain the sensitivity of the assay.
Kyo et al. (2005) Anal.Chem. 77: 7115
Cyclic voltammetry and electrochemical impedence spectroscopy Anti-PSA immobilized on gold electrode, PSA analyte
Fragoso, A., Laboria, N., Latta, D., and O’Sullivan, C. K. (2008) Anal. Chem. 80, 2556-2563
Anti-BSA Binding to Immobilized BSA using SPT-0013/SPT-0014A mixed dithiol SAM
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17 nM
8.3 nM
4.2 nM
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0.52 nM
0.26 nM
0.13 nM
0.06 nM0.03 nM
T. Ryan, Reichert,Inc
Surface Plasmon Resonance Test for Non-Specific Binding to Bidentate (dithiol) SAM Derivatized with BSA Antigen vs a
1,000-fold excess of nonspecific antibody ���
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20.5 ng/mL Anti-BSA20 ug/mL Sheep IgG SPT-0013/14
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T.Ryan, Reichert, Inc
SPR: Reproducibility
10 injections each of 13.3 nM, 4.43 nM and 1.47 nM Anti-BSA IgG over very low density (150 uRIU) BSA surface
T. Ryan, Reichert, Inc
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SPT-0013/14
96 Consecutive Anti-BSA IgG Injections (13.3, 4.43, 1.47 nM) over immobilized BSA on dithiol mixed SAM Each antibody injection was followed by a regeneration injection (20 mM HCl) and a buffer injection (PBST), Total 288 Injections
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SPR: Stability
T. Ryan, Reichert, Inc.
SPT-0013/14 O
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Slide Functionalization for Surface Plasmon Resonance Study of Mannose Binding
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Mannose- Derivatized SAM
Mixed Dithiol SAM
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Schlick,KH and Cloninger, MJ Tetrahedron 66(2010)5305-5310
Extreme Stability Biacore SPR
SPT-0013/SPT-0014A
Biacore Surface Plasmon Resonance Measurements of Saturation Binding
• Experimental Method: – Serial dilutions of protein are
added over the surface – Signal is measured with protein
additions – Rate and equilibrium constants
are determined on SAME SLIDE REPEATEDLY OVER A PERIOD OF SEVERAL MONTHS
Au Con A
Con ACon A
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Laminar flow
Equilibrium constants are determined Rate constants are determined
Schlick,KH and Cloninger, MJ Tetrahedron 66(2010)5305-5310
SPR: Sensitivity
Binding of Ins-mAb at 20 g/ml in the presence of insulin at various concentrations (0–300 ng/mL) over the insulin immobilized rigid-rod OEG-DCA SAM chip. The same sensor chip was used for all the experiments with surface regeneration cycles using glycine·HCl buffer injection.
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SPT-0005
Gobi, et al.(2007) Biosensors & Bioelectronics 22:1382
Gold Nanoparticles
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In vivo Imaging: Specificity
Darkfield reflectance imaging of live NIH3T3 cells labeled with varying 20 nm gold nanoparticle contract agent formulations: (a) unlabeled cells; (b) Np not functionalized (c) Np functionalized with anti-actin targeting
antibody; (d) Np functionalized with only delivery peptide; (e) Multifunctional contrast agent with both
targeting and delivery moieties. Arrows indicate the presence of visible
filapodia.
SPT-0014B-hydrazide
SPT-0014B linker
Kumar et al. (2007) Nano Lett 7:1338
20nm GNP
50nm; -‐10mV
Targe3ng mAb
An3-‐bio3n mAb
PEG
Poly-‐GL
Bio3n
10BSH
Dithiol-‐PEG-‐hydrazine Linker SPT-‐0014B
Nanoparticle Platform for Targeted Boron Neutron Capture Therapy
Webb, A.C., Choi, R., Sun, S, Flynn, N., Kolodny, N.H. & Riley, K.(2012). Resurrection of boron neutron capture therapy in an era of nano-oncology. 20th Ann. Cancer Res. Inst. Internat.Symp.Cancer Immunotherapy 2012. New York, NY. Oct. 1-3.
Principle of force curve measurement (a) approach, (b) contact with surface and ab-ag binding, (c) pull out, and (d) pull further until unbinding of ab-ag is achieved. An example force curve (left). It took ~100 pN force to break the ab-ag binding of collagen.
Collagen antibody-antigen
Avci, R. & Suo Z. Montana State University
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Step1: Peg6 tip & bare mica Step2: Peg6 tip & mica with Cytochrome c Step3: Unfolding of Cytochrome c
Examples of force curves taken by an AFM tip functionalized with PEG6 molecules, and immersed in a TRIS buffer at pH 8: (a) Clean mica in TRIS, (b) 100 µL mixture of 25 µg/mL cytochrome C (CC) in TRIS, and (c) subsequent force-distance curves on clean mica surface in TRIS. The observed pull-off event (~100 pN magnitude) is due to unfolding of a CC protein stuck on the AFM tip. Attachment of CC to the AFM tip reduces drastically with increasing PEG length.
Avci, R. & Suo, Z. Montana State University
Benzaldehyde-PEGn-NHS PEGylated Linkers for Single Molecule AFM
n= 6,21,76,100 Eg units
MW 264,1000,3500,5000
SPT-6,21,76,100
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C61H107NO29Mol. Wt.: 1318.49
For PEG 21-22 (PEG1000) ESTIMATEDtotal extended length = 97.8-98.8 ÅPEG 22 extended length = 92.8-93.8 ÅPEG length = 3.9-4.9 Å (4.2 Å calc)
For PEG 76-77 (PEG3400) ESTIMATEDgiven 4.2 Å/PEG = 319,2-323.4 Åadd 5.25 Å for terminiTotal estimated extended length = 326 Å
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OMW 142
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MW 119
Est MW ~1000+260
Est MW ~3350-3400 + 260est C169H323NO83
3.9-4.9 Å
92.8-93.8 Å
97.8-98.8 Å
Biosensors built from gold film to measure exquisitely small molecular interactions are critical analytical tools for drug development, diagnostics, and fundamental research applications.
SUMMARY Linkers and Tethers
Surface plasmon resonance (SPR), in single and multiplexed array formats, quartz crystal microbalances (QCM), atomic force microscopy (AFM), electrochemical assays, and in vivo imaging using gold nanoparticles comprise a large and expanding market that relies on unique and effective surface chemistries to evaluate macromolecular and single molecule interactions
Each of these methods depends directly on the acquisition of a target analyte by a covalently immobilized capture ligand.
The immobilization is accomplished by a heterobifunctional tether or linker adsorbed through a thiol to the sensor’s gold surface, and a terminal functional group for covalent coupling by a variety of chemistries to proteins or DNA. The system lends itself to controlled ligand loading enabled by selecting the percentage of functionalized tether in the mixed SAM
Alkanethiolates interacting with a planar gold surface tend to desorb leaving bare gold patches available for non-specific adsorption of proteins thus compromising data analysis.
Since polyvalent molecular interactions are one of the basic concepts in biological recognition, we have developed a series of bidentate dithiol pegylated dendron linkers, used together with a pegylated background dendron.and functionalized linkers, with consequent high levels of stability on the gold film and excellent resistance to non-specific binding or sensor fouling, leading to enhanced selectivity, specificity and sensitivity, since the tethered capture ligand is not lost in a fluidic environment.
The result is a flexible, stable, reliable surface chemistry that is adaptable to most types of sensor instrumentation.