Resonating nanomechanical microcantilevers for quantitative biological measurements in liquid

Nanomechanical cantilevers have been proved as extremely sensitive time resolved mass
and surface stress sensors. Recent demonstration of zepto gram (10−21 grams) sensitivity
in vacuum and label free detection of human RNA biomarkers in the total background
of cellular RNA at pM range are the unprecedented achievements with these sensors.
However, using them as mass sensors in liquid has always been a great challenge. This
thesis describes the successful attempt in resonating microcantilevers in liquid and using
them as quantitative mass sensors for biological applications. Futhermore, simultaneous
measurement of mass sensing and surface stress sensing provides a unique system which
can detect multistep and multiprocess systems in biology without any labels.
The Instrument:
Chapter 1 describes the present status of these sensors in various fields. A review of
interesting experiments performed by various researchers is presented. In Chapter 2
a detailed description of the setup which uses a tiny liquid chamber of 6 μl to hold a
microcantilever array is given. Working principles of various parts and mathematical
description of basic operating modes are also discussed. Importance of laser beam
diameter for detection and a model test result of simultaneous stress and mass sensing
are given in the results section.
Higher modes of vibration increase mass sensitivity:
Operating the microcantilevers at higher modes increases mass sensitivity by at least
two orders of magnitude. This is demonstrated by uniformly deposited gold layers on
the cantilevers (see Chapter 3). A sensitivity increase of 940 ag/Hz/μm2 at mode 1
to 8.6 ag/Hz/μm2 at mode 7 under ambient conditions has been proved. The limitation
of mass load with cantilever thickness is dealt in detail.
Resonating microcantilevers in liquid:
In Chapter 4, a frequency spectrum with clear well resolved resonance peaks for 16
flexural modes of vibration in liquid is presented. The increase in quality factor with
mode number is shown. The predicted frequency values from theoretical models and
the estimated added apparent mass due to liquid on the resonating cantilever at various
modes are compared. The independent effect of density and viscosity at various modes
of vibration is discussed in Chapter 5. A distinguishable difference between peak
frequency and eigen frequency is revealed in the plots.
Mass measurement of latex beads in liquid:
A test system based on binding of biotin labeled latex beads to the streptavidin functionalized
cantilever for measurement of mass sensitivity is described in Chapter 6. The
increase in mass sensitivity at higher modes even in liquids is clearly demonstrated. A
total of 7 ng was detected with a resolution of 1 ng.
Quantitative biological measurements:
In Chapter 7 quantitative measurement on real time biological system is presented. A
new functionalization technique based on inkjet spotting was used to immobilize 2D
crystals of reconstituted FhuA transmembrane proteins. FhuA functionalized cantilevers
were found sensitive to detect the binding of ligands T5 phage virus particles (10−17g)
and ferrichrome molecules (700 Da).
Detecting multistep and multivariant biological system:
An experiment demonstrating the capability of the system to perform simultaneous mass
sensing and stress sensing is described in Chapter 8. The mass measurement plots
reveal that only monolayer formation of lipid vesicles occurs. A multistep experiment
with initial vesicle adsorption and subsequent binding of bee venom protein (melittin)
demonstrates measurements in different mass ranges. Simultaneously, tensile stress on
the cantilever due to vesicle adsorption and the subsequent compressive stress owing to
the pore formation of melittin in the immobilized vesicles are observed in the deflection
plot. Finally Chapter 9 concludes summarizing all the results obtained.