# Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?
## 4 Different Types of Tethered-Bilayer Lipid Membranes
### Sparsely-tBLMs or stBLMs
Finally, stBLMs have been used as sensing elements of biochips for the detection of lethal factor (LF) secreted by _Bacillus anthracis_, after self-insertion of _Bacillus anthracis_ PA63 ion channels, an octameric pore which bonds LF, in preformed stBLMs [152]. [PERSON] et al. [87] have developed a versatile and simple \"bottom-up\" approach to create stBLMs on the coating of oxidized surfaces of most nanoelectronic devices, using a covalent tether attachment chemistry based on silane functionalization, followed by step-by-step stacking of two other functional molecular building blocks, oligo-PEG and phospholipid (i.e., DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). In this process, oligo-PEG serves as both hydrophilic spacer and tether for the grafting of DOPE; standard vesicle fusion process was used to complete the bilayer formation. These stBLMs have been formed on three common classes of (nano)electronic biosensor devices (i.e., indium-tin oxide-coated glass, silicon nanoribbon devices, and high-density single-walled carbon nanotubes (SWNT) networks on glass). The fluidity of the stBLMs has been checked by FRAP and _alamethicin_ has been used to record real-time single ion channel activities with a high sensitivity and spatial resolution, demonstrating the successful integration of nanoelectronic devices with electrophysiology [87]. The need for a diluted sub-membrane space and a consequential increase in sub-membrane hydration has also been demonstrated using a cholesterol-tethered membrane with 6-mercaptoethanol (6 MH) acting as a diluting agent [155; 157]. In this case, the anchorlipid is also constituted of three structural elements: a thiol group, as the surface chemical linker, connected to a triethylene glycol as hydrophilic spacer unit, itself linked to cholesterol as membrane lipid anchor (i.e., ethyleneoxy-linked cholesterol or EO\({}_{3}\)C). Membrane formed with a mixture of anchorlipid EO\({}_{3}\)C and 6 MH have revealed good electrical resistance properties, and hydration of the sub-membrane compartment has been ascribed to a phase separation creating nanodomains of 6 MH allowing formation of spacious aqueous reservoirs (large spaces) underneath the bilayer tethered by the ancholipid EO\({}_{3}\)C (Figure 1) [82]. EO\({}_{3}\)-cholesterol anchorlipid and derivatives have been successfully used to study transmembrane transport mediated by small peptide ionophores like _valinomycin_ or _gramicidin_[156; 157; 182], to scrutinize the conformational changes during the transition from the \"open\" to the \"closed\" state of the human transmembrane voltage-dependent anion channel (hVDAC) [108], or to investigate the enzyme activity of transmembrane proteins like _cytochrome bo\({}_{3}\)_, an ubiquinol oxidase [109; 119; 183; 184] or _CymA_, a menaquinol-7 dehydrogenase of _Shewanella oneidensis_[185], all functionally reinserted into cholesterol-stBLMs by using proteoliposome fusion to form the distal layer. Cholesterol anchored bilayers have also been used to create an in vitro mimic of the cytoskeletal scaffold after reinsertion of _ponculin_, a protein of mold (_Dictyostelium discoideum_), able to bind F-actin network of cytoskeleton and serving as nucleation site for actin polymerization [186]. These membrane models were useful to investigate interaction of antibiotics, like _vanomycin_ or _ramoplanin_, with stBLMs inserting two precursors to peptidoglycan formation in order to mimic the membrane of Gram-positive bacteria for addressing questions related to antibiotic action and resistance [187]. Finally, sparsely cholesterol anchored bilayers have been successfully used to scrutinize the process by which _alamethicin_, an antimicrobial peptide, absorbs to and then integrates into a lipid bilayer. _Alamethicin_ acts by assembling in pores made up of four to six individual peptide strands.