Page 57 - Nanotechnology an introduction

related to well-established ones—indeed metrology was ably handling measurements in the nanoscale long before the term “nanotechnology” was
introduced and even before Feynman's lecture [56]—when living organisms are introduced into the system it is moved into unfamiliar territory.
Particular problems are associated with: the very narrow range of ambient conditions in which life can be maintained (otherwise the life must be
terminated and the sample fixed, but then one may not be measuring what one wishes to know); the fact that life is mainly composed of soft,
irregular structures that are constantly evolving on different timescales; and the fact that any living organism, even a single cell, is able to modify its
environment (e.g., by secreting macromolecular substances).

One of the most important techniques yielding nanometer resolution is electron microscopy (EM, Section 5.1.2). In principle, however, this is ruled
out for investigating the nano/bio interface because of the damage to delicate biostructures caused by the necessary sample preparation, typically
including dehydration and impregnation with heavy metals [74]. Given that water is the dominant molecular species in nearly every biological
system, the ability to operate in an aqueous environment is an essential prerequisite.
Some measurement problems associated with nanotechnology may not require nanometrology. For example, the toxicologist investigating the
effect of nanoparticles on a living organism may merely administer the nanoparticles intravenously and observe the subsequent behavior of the
animal.

5.5.1. Determining the Nanostructure of Protein Coronas

The protein corona is the collection of proteins that accumulate on an artificial surface introduced into a biofluid such as blood; that it, the process of
opsonization. Since there are many hundreds of different proteins (far more if one includes all the IgG antibodies of different affinities as “different
proteins”) it is very likely that the surface becomes coated with a complex structure comprised of many different proteins and, moreover, constantly
changing with time.

A major goal is to be able to design surfaces that can determine the nature of the corona. To achieve this, one must understand how the corona is
assembled. Unlike other self-assembly problems, the corona is constantly changing its composition and structure. The main challenges are
identifying the proteins and characterizing the structure. The first of these is very difficult. If one exposes the corona-decorated nano-objects to a
series of different monoclonal antibodies, this would allow one to at least identify the outermost proteins, but the procedure becomes impracticable
for more than a few proteins; apart from the expense of procuring the monoclonal antibodies, the initial ones to bind will block access by the
subsequent ones. It must, moreover, be repeated over a long succession of intervals. On the other hand, the reflectance techniques described in
Section 5.3 are well applicable to measuring the nanostructural evolution of the corona in situ. OWLS in particular is well able to handle anisotropy
[79] and refractive index profile determination [115], as well as overall thickness and mean refractive index of the corona.

5.5.2. Measuring Cell Adhesion: the Interaction of an Evanescent Field with a Cell

The evanescent field decays exponentially perpendicular to the plane of the waveguide (see, e.g., [162]), and the characteristic decay length 1/s (in
which the field intensity decays to 1/e of its value at the waveguide surface), also called the penetration depth, depends on the refractive indices of
the waveguide, the waveguide support S, the cover medium C (including the cells), and the film thickness d (see Figure 5.5). For the transverse
F
electric (TE) guided lightmodes the inverse penetration depth is given by

(5.22)
where k is the wave vector of the light (numerically equal to 2π/λ, λ being the vacuum wavelength), N is the effective refractive index of the guided
mode, and n is the refractive index of the composite cover medium (culture fluid and cells) as sensed by the guided lightmode. This is for decay
C
into the cover medium; the inverse decay length into the substratum is

(5.23)
In using these equations one should bear in mind that N also depends on n as well as all the other waveguide parameters.
S
To a first approximation, we can suppose that at any distance z from the waveguide surface, the cover medium refractive index is the arithmetic
mean of that of the cells and that of the culture fluid:

(5.24)
where n is the refractive index of the cell material (which may itself depend on z), c is the number of cells per unit area of the waveguide, A(z) is the
κ
cross-sectional area of the cell parallel to the waveguide at a distance z from the waveguide surface, and n is the refractive index of the pure
M
culture fluid. Hence, to determine the total contribution of the cells to n , we need to integrate the product A(z)e −sz from z = 0 to ∞; this is just the
C
Laplace transform of A(z), i.e.,

(5.25)

v′ is called the effective volume of the cell [144].
By taking the Laplace transform of the right-hand side of equation (5.24) and dividing by the total volume effectively sensed by the waveguide,

equal to , we obtain the final expression for the cover refractive index:

(5.26)
where we have assumed that n is the refractive index throughout the cell. If this is considered to be inadmissible, for example when taking into
κ