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Page 1

Mechanochemical study of single living

cells and biomolecules combining

optical tweezers and Raman spectroscopy









Saurabh Raj











ICFO – The Institute Photonic Sciences

Universitat Politècnica de Catalunya

Barcelona 2012

Page 73

Detection of single DNA molecule in optical trap using SERS 49

and our setting of the DNA at about 70 % extension before measuring Raman
signal combined to minimize this �uctuation, as could be seen in the live 1 s
acquisition scans.

To verify that the Raman signals originate from a single DNA molecule,
force-extension curves [82] were measured of the DNA molecules that produced
a Raman signal. Fitting the curves in Fig. 5.2 to the well established worm-
like chain (WLC) model [83] allows two basic parameters to be extracted: the
contour length (L0) and persistence length (P ) (formula given in Fig. 5.2a inset).
In all measurements, the measured contour length, at 4.1 µm, was consistent and
veri�ed our ampli�cation protocol for this length and the rough length estimate
found with electrophoresis.

The �nal con�rmation of a single DNA molecule between the beads should
be the extension curve �tting to the DNA natural persistence length of 53
nm [82,84]. This value would change proportionally to the number of molecules
being extended. In the sample set, the persistence length values ranged be-
tween 47-57 nm which is within reasonable error of the well-established value
for a single DNA molecule. Most likely due to the metal particles, the �ts are
not as good as what is typically seen for these types of measurements. Thus,
in order to ensure that the curves are beyond a reasonable error range from a
multi-molecule ensemble, theoretical force-extensions curves for two persistence
lengths, 53 nm and 26 nm, are included in Fig. 5.2 with the experimental data
and original �t (where all parameters were allowed to vary). The lower persis-
tence length estimates the e�ective rigidity of two DNA molecules which forces a
large deviation of the WLC model from the experimental data. However, the 53
nm value for a single DNA molecule is extremely consistent, not even changing
more than 5% at di�erent pH conditions [82]. This leads to our conclusion that
although our measured values indicate a single DNA molecule, the mechanics
of the DNA are being a�ected by the attached metal particles.

The WLC �ts are weakened by the higher force values in the region between
80% (3.3 µm) and 88% (3.6 µm) extension or 0.5 - 1.5 pN. The region is rep-
resentative of the DNA transitioning from entropic dominated forces to acting
like a structure with an intrinsic elasticity [85]. The attached metal particles
do not seem to a�ect the entropic forces that are necessary to straighten the
DNA nor the linear elastic region at higher forces. The average diameter of the
silver particles is larger than the persistence length of DNA. Thus, if there are
clusters of few Ag particles, the DNA could tend to coil around the metal rather
than taking on a rod-like behavior with just one particle. The force needed to
uncoil these segments would certainly occur at low values, because of the non-
speci�c binding, but should be greater than what is needed to straighten the
DNA to a rod at normal aqueous and thermal conditions. The absence of an
e�ect at longer extension, or larger forces, is also due to the size of the metal
particles, but this time in an opposite way. The nanometer sized diameters of
the metal particle or clusters are much less than the microns length scale of
the DNA, leaving stretching forces in the length direction una�ected when the
DNA is acting as a rod with a �nite stretch modulus. This is also dependent
on the low density of metal on the DNA molecule, which is con�rmed from the
concentrations used.

This technique of the single molecule DNA detection o�ers distinct advan-
tages over the current methods of studying single DNA spectroscopically that
require the DNA to be stuck to a surface in order to realize the reproducibility

Page 74

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Detection of single DNA molecule in optical trap using SERS 50

2500 3000 3500 4000
0

1

2

3

4

2500 3000 3500 4000
0

1

2

3

4

5



F
or

ce
F

(
pN

)

Length x (nm)

b)



F
or

ce
F

(
pN

)


Length x (nm)

a)

Figure 5.2: A sample of two extension curves from the data set of DNA molecules
that gave a Raman signal at the conditions of Fig. 5.1. The curves are �t to the
WLC model (red line) (formula in (a) inset) and all curves give the expected contour
length (L0) of 4.1 µm and persistence lengths (P ) that vary between 47 - 57 nm. In
the two examples given here, persistence values of (a) 55 nm and (b) 48 nm were
found. Error bars are calculated to be far less than the size of the plotting symbols
and are therefore not shown. Theoretical force-extension curves are included using the
expected contour length and persistence lengths of 53 nm (blue line-square) and 26
nm (green line-triangle) in order to further demonstrate the presence of a single DNA
molecule. A camera image of the suspended DNA-bead construct is included in (b)
inset.

Page 145

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