269
experimental factors influence SSHG spectroscopy results. These include the pulse
width, average power, wavelength and polarization of the laser, the numerical aperture of the microscope objective, the length of interaction, the detection geometry of
the imaging system, as well as inter-dependences between these factors [3].
Moreover, one of identified flaws in this study was complication introduced by random immobilisation of biomolecules. This is in agreement with other workers [4-5].
who identified the problem facing implementing optical biosensing techniques as
difficulty of obtaining a reproducible, well-defined sensor surface, due to random
immobilisation of biomolecules. Thus, it is more likely that recorded SSH signals
during each stage were attributed to the aggregate effect of bioassays including
sources within the microwell materials [6-7]. So to ensure reproducibility within the
data, experiments should be designed on media or substrates with uniform SSHG
characteristics. Also this phase of the study indicated clearly that there is a need to
record SSHG data from different stages of assay preparation. This would enable
monitoring of SSHG susceptibility introduced by each stage. It is worth noting that
in an optimal configuration, it would be expected that most of protein molecules in
the upper monolayer would contribute to the signal sensor. Therefore it would be
very interesting to experiment with diversified surfaces that would produce anisotropic and oriented immobilisation of protein. The use of diversified surfaces that
Fig. 28.4 Residuals and
cumulative sum (cusum) of
Passing and Bablok
regression analysis for
comparing ELISA Optical
Density to ∆SSHG interact of
the SSHG spectroscopy
28 Evaluation of Surface Second Harmonic Generation SSHG for Detecti…
experimental factors influence SSHG spectroscopy results. These include the pulse
width, average power, wavelength and polarization of the laser, the numerical aperture of the microscope objective, the length of interaction, the detection geometry of
the imaging system, as well as inter-dependences between these factors [3].
Moreover, one of identified flaws in this study was complication introduced by random immobilisation of biomolecules. This is in agreement with other workers [4-5].
who identified the problem facing implementing optical biosensing techniques as
difficulty of obtaining a reproducible, well-defined sensor surface, due to random
immobilisation of biomolecules. Thus, it is more likely that recorded SSH signals
during each stage were attributed to the aggregate effect of bioassays including
sources within the microwell materials [6-7]. So to ensure reproducibility within the
data, experiments should be designed on media or substrates with uniform SSHG
characteristics. Also this phase of the study indicated clearly that there is a need to
record SSHG data from different stages of assay preparation. This would enable
monitoring of SSHG susceptibility introduced by each stage. It is worth noting that
in an optimal configuration, it would be expected that most of protein molecules in
the upper monolayer would contribute to the signal sensor. Therefore it would be
very interesting to experiment with diversified surfaces that would produce anisotropic and oriented immobilisation of protein. The use of diversified surfaces that
Fig. 28.4 Residuals and
cumulative sum (cusum) of
Passing and Bablok
regression analysis for
comparing ELISA Optical
Density to ∆SSHG interact of
the SSHG spectroscopy
28 Evaluation of Surface Second Harmonic Generation SSHG for Detecti…