BioInterface Flashcards
What is a biointerface?
The interface between the BioMEMS device (cantilever) and the analyte (aqueous environment)
What are some examples of BREs?
Antibodies (pAb/mAb), Receptors, peptide aptamers, DNA/RNA aptamers
What are the advantages of using pAbs and mAbs Abs as BREs on cantilevers?
pAbs - bind multiple epitopes on antigen - not specific - useful to increase signal from antigen binding, when we don’t know the specific antigen, not affected by changes in antigen
mAbs - bind specific antigen - very specific - reduces background noise and cross reactivity
What is the Kd?
Disocciation constant - determines the affinity of binding of analyte to the receptor - ideally between nm and pm.
How do we attach a BRE to the cantilever?
Use of a SAM - alkanethiol head group, aliphatic hydrophobic hydrocarbon chain, functional head group
Spontaneous covalent bond forms between sulphur group in SH and gold on cantilever
What are the two ways to bind BRE to the functional head group?
Physisorption
Chemisorption
How does physisorption work?
change in pH causes Nh2 to go to Nh3+, this can attract negatively charged molecules - cheap/fast/easy
Negatives - physical contact with surface causes unfolding of protein - orientation not specific - pH change can cause leeching
How does chemisorption work?
Lysine group in protein attacks positive carbon centre of COOH, condensation reaction, forms strong amide bond (covalent)
Pos - much stronger, won’t come into contact with surface - not affected by pH
Neg - orientation still random
How can overcome the idea of random orientation in chemisorption?
Orientated chemisorption - inclusion of a cysteine group (by recombinant cloning) - works for antibodies, receptors and peptide aptamers - relies on no other cysteine residues in molecule
How can we produce an optimal surface density? Why is it important that we do?
Combination of blocking (PEG) and binding monolayer - too many binding SAMs can lead to steric interference and lead to reduced BRE immoblisation
What can affect binding of SAM to a cantilever?
Dirty/impurities on surface
pH
What are the methods of site specific immobilisation?
Microcontact printing
Microcapillaries
Microspotter
Dip pen nano lithography
How does micro contact printing work?
Produce MASTER MOULD using micro fabrication - coat with PDMS - produce PDMS stamp - dip into ink (alkanethiol INK or protein/DNA/Ab) - bring into contact with gold surface - ink that is in contact will be transferred in specific pattern
What must be do when performing micro contact printing with biomolecules (e.g. proteins)?
Make PDMS stamp hydrophillic
What are the benefits of the PDMS stamp being soft?
Wont damage the ink or the substrate
