248 6.6  Electrical Force Tools

However, none of these modifications is ideal as they all affect the native cell physiology.

The use of smaller diameter pipettes is a less perturbative improvement—​as for SICM, glass

micropipettes may be heated and controllably stretched to generate inner diameters down to

a few tens of nanometers. Ion channel current measurements may also be performed in com­

bination with fluorescence imaging—​if a fluorescence maker can be placed on a component

of the nanopore, then it may be possible to count how many ion channels are present in the

patch clamp region directly, through controllably placing a fluorescent tag on a nanopore but

avoiding impairment of the ion channel function is nontrivial.

Many researchers also utilize similar electrophysiology techniques on larger tissue

samples. The most popular biological systems to study involve muscle and nerve tissue.

Much of the early historical research involving biophysical techniques used electro­

physiology approaches, but many of these methods are still relevant today. In essence,

they involve either excised tissue or, as is sometimes the case for cardiac muscle studies,

experiments using whole living animal models. Electrodes are relatively large in length

scale compared to the thinned micropipettes used for patch clamp methods, for example,

consisting of metal needles or micron length scale diameter micropipettes filled with elec­

trolyte solution.

Although lacking some of the finesse of patch clamping, traditional electrophysiology

methods have a distinct advantage in generating experimental data in a physiologically rele­

vant tissue level environment. The importance of this is that single cells respond electrically

to both chemical and mechanical triggers of their neighbors in addition to their intrinsic elec­

trical properties at the single-​cell level. These effects are very important in the emergence of

larger length scale properties of whole tissues, for example, in determining the complicated

beating rhythms of a whole heart. There is also significant scope for valuable biophysical

modeling of these complex whole tissue electrical events, and the cross length scale features

are often best encapsulated in systems biophysics approaches (i.e., systems biology in the con­

text of biophysical methodology), which are discussed in Chapter 9.

6.6.3  SOLID-​STATE NANOPORES

Modern nanofabrication methods now make it possible to reproducibly manufacture

nanopores using synthetic silicon-​based solid-​state substrate. One popular method to

manufacture these involves focused ion beam (FIB) technology. FIB devices share many

similarities to TEMs in generating a high-​intensity beam of electrons on the sample. The

beam is focused onto a thin sheet consisting of silicon nitride, which generates a hole. By

varying the power of the beam the size of the nanopore can be tuned, resulting in repro­

ducible pore diameters as low as ~5 nm (van den Hout et al., 2010). Such nanopores have

been applied successfully in the detection of single molecules of a variety of biopolymers

including nucleic acids (Rhee and Burns, 2006) and also have been used to measure the

unfolding macromolecules.

Molecular detection using ion flux through solid-​state nanopores involves first applying a

voltage across either side of the nanopore, which causes ion flow through the pore in the case

of a typical physiological solution. However, any biopolymer molecules in the solution will in

general possess a nonzero net charge due to the presence of charges on the molecular surface,

resulting in the whole molecule migrating down the voltage gradient. Due to the large size

of biopolymer molecules, their drift speed down the voltage gradient will be much slower

than that of the ion flow through the nanopore. When a biopolymer molecule approaches

the nanopore, the flow of ions is impeded, maximally as the molecule passes through the

nanopore. The drop in ion current is experimentally measurable if the translocation speed

through the nanopore is sufficiently slow. The specific shape of the drop in current with time

during this translocation is a signature for that specific type of molecule, and so can be used

as a method of single-​molecule detection.

With greater spatial precision than is currently possible, a hope is to consistently measure

different nucleotide bases of nucleic acids as a single molecule of DNA migrates through

the nanopore, hence sequencing a single DNA molecule rapidly. The main problem with