Ultrahigh Surface Sensitivity of Deposited Gold Nanorod

Arrays for Nanoplasmonic Biosensing

Group No.4
Name Roll No
Maheen Riasat 232659
Anusha Khurram 232660
Anum Abbasi 232656
Javeria Bibi 232662
Submitted To
Dr. Waqas Khalid
Electrodynamics

Department of Physics
Date: Dec26th, 2023
Contents
Introduction....................................................................................................................................3
Materials and Method...................................................................................................................3
UV-Visible Spectroscopy Measurements....................................................................................3
Bulk refractive index sensitivity measurement of AuNRs (gold Nano-rods) in solutions..........4
Sensor chip fabrication.................................................................................................................4
Scanning electron microscopy imaging.......................................................................................4
Localized surface plasmon resonance (LSPR) measurement......................................................4
Bulk refractive index sensitivity measurements of deposited AuNRs.........................................4
Results and Discussion..................................................................................................................5
Nanoplasmonic sensing strategy..................................................................................................5
Plasmonic characterization..........................................................................................................5
Bulk sensitivity measurements.....................................................................................................5
Surface sensitivity evaluation......................................................................................................6
Biosensing performance comparison...........................................................................................6
Conclusion......................................................................................................................................6
Introduction

Nanoplasmonic sensors offer high sensitivity, simple instrumentation, and label-free readout for
tracking bio macromolecular interactions at solid-liquid interfaces. As sensor development
evolves, performance metrics are crucial for understanding plasmonic behavior. Light extinction
in metallic nanostructures causes the oscillation of conduction-band electrons, resulting in a
plasmon-enhanced electromagnetic field. This effect, known as localized surface plasmon
resonance (LSPR), is typically 5-20 nm long and changes due to bio macromolecular interaction
events in metallic nanoparticles. Light extinction occurs across UV-visible wavelengths, with a
maximum at λmax for each plasmon mode. Nanoplasmonic sensors design for biosensing
applications should elicit larger λmax shifts for target bio macromolecular interactions, for
improved detection sensitivity, accuracy, and reliability. Design strategies for plasmonic
nanostructures include larger field enhancements and shorter decay lengths, with rational tuning
based on performance criteria. The bulk refractive index sensitivity is the primary criterion for
evaluating nanoplasmonic sensor performance, describing the measurement response to changes
in the refractive index above the sensor surface. This approach, adapted from conventional
surface plasmon resonance biosensors, is also crucial for nanoplasmonic sensors with shorter
decay lengths. Surface sensitivity evaluation focuses on changes in the local refractive index near
the sensor surface, representing biosensing measurements. Approaches include atomic layer
deposition of thin dielectric layers or adsorption of bio macromolecule models. Comprehensive
evaluations have been performed for laterally isotropic nanostructures, but there is a need to
investigate laterally anisotropic nanostructures like nanorods for high-performance
nanoplasmonic sensing platforms.

The study presents the creation of plasmonic gold nanorod arrays (AuNR) with high surface
sensitivity for detecting bio macromolecular interactions. These arrays use transmission-mode
LSPR sensing, high temporal resolution, and real-time surface assimilation. The LSPR-based
measurement readout relies on AuNR direction, instrumentation, and surface coverage.

Materials and Method

UV-Visible Spectroscopy Measurements
AuNRs (gold Nano-rods) are nano scale particles that have rod like structure and exhibits strong
optical absorption in the near infrared region due to their localized surface plasmon resonance
(LSPR) effects. The (LSPR) of AuNRs depends on their size, shape aspects ratio and
surrounding medium. By tuning these perimeters it can be used for various applications such as
photothermal therapy, biosensing, catalysis and imaging.
To characterize the plasmonic properties of AuNRs, UV visible spectroscopy is a common
technique that measure the absorption spectrum of nanoparticles in solution. The absorption
spectrum of AuNRs (gold Nano-rods) typically shows two peaks; the one corresponds to the
transverse plasmon mode and other to longitudinal plasmon mode. The TEM is usually located
in the visible region and is relatively insensitive to the aspect ratio of the Nano-rods while (LPM)
is located in the NIR region and shifts with the aspects ratio and the reflective index of medium.
By measuring the absorption spectrum of AuNRs across 300-1000nm wavelength range one can
determine the plasmon peak position. The aspects ratio and the extinction coefficient of the
Nano-rods. These parameters can be used to evaluate the quality, stability and functionality of
the AuNRs for different purpose.
Bulk Refractive Index Sensitivity Measurement of AuNRs (Gold Nano-rods) in Solutions
The process of diluting the stock AuNR solution by half in water/glycerol mixtures with
increasing glycerol fraction to obtain aqueous AuNR dispersions containing 0–35% v/v glycerol
in 5% increments. The extinction spectra of the AuNR dispersions were measured using the UV–
vis spectrophotometer and the longitudinal peak shifts were plotted against the change in
refractive index of the solution. The gradient of the plot was determined by linear regression
analysis and corresponded to the bulk refractive index sensitivity of the solution-phase AuNRs.
Sensor Chip Fabrication
To prepare glass substrate coated with gold Nano particles using chemical linkers. Firstly Blank
glass substrates were sequentially cleaned with 1% SDS (removing oil strains and residues from
glass substrate), deionized water, and ethanol, followed by drying under a stream of nitrogen gas.
The substrates were then subjected to oxygen plasma treatment for 1 min prior to static
incubation in a solution of 10% (v/v) APTES in ethanol for 30 min. After that the sensors were
extensively washed with ethanol and dried under a stream of nitrogen gas. The APTES-coated
substrates were then treated in an oven at 110 °C for 1h, before cooling down to room
temperature. The APTES-coated substrates (a process of attaching APTES molecule to oxide
surfaces such as glass, silica) were incubated in a AuNRs solution for 3h after which they were
washed with deionized water and finally dried under a stream of nitrogen gas.
Scanning Electron Microscopy Imaging
The Instrument used a JEOL JSM-6700 field emission scanning electron microscope to study
the surface coverage of gold nanorods (AuNRs) on glass substrates. Method used to directly
imaged the AuNR-coated glass substrates at a voltage of 5.0 KV and calculate the surface area
coverage using the ImageJ software.
Localized Surface Plasmon Resonance (LSPR) Measurement
The Instrument used an Insplorion XNano instrument to conduct transmission-mode LSPR
experiments. The as-fabricated sensor chips to oxygen plasma treatment for 30 seconds before
loading them into the microfluidic chamber. They used a Reglo Digital peristaltic pump to inject
liquid samples into the microfluidic chamber at a flow rate of 100 μL/min. The LSPR peak
wavelength from the longitudinal plasmon mode in the optical extinction spectrum was
determined by high-order polynomial fitting. The measurement data were collected with a time
resolution of 1 Hz, and data analysis was performed using the Insplorer software
package (Insplorion AB). The authors used a high-order polynomial fitting to determine the
LSPR peak wavelength from the longitudinal plasmon mode in the optical extinction spectrum.
Bulk Refractive Index Sensitivity Measurements of Deposited AuNRs
The signal response of gold nanorod (AuNR)-coated glass substrates to different water/glycerol
mixtures using the localized surface plasmon resonance (LSPR) technique. They injected the
mixtures into a microfluidic chamber and tracked the shifts in the peak wavelength from
the longitudinal plasmon mode in the optical extinction spectra. They plotted the peak shifts
against the change in refractive index of the solution and calculated the gradient of the
plot by linear regression analysis, which represented the bulk refractive index sensitivity of
deposited AuNRs.
Results and Discussion
Nanoplasmonic Sensing Strategy
The strategy for designing and testing a nanoplasmonic sensing platform using gold nanorods
(AuNRs). Two types of AuNRs were employed short with a length of 48 nm and an aspect ratio
of 2.7 and long with a length of 55 nm and an aspect ratio of 3.6. The AuNRs were deposited on
an APTES-functionalized glass substrate, resulting in distinct plasmonic properties visible as
blue-greenish and pale purplish tints.
Scanning electron microscopy confirmed well-separated AuNRs with low surface coverage.
Plasmonic properties were characterized through refractive index sensitivity measurements and
theoretical analyses. Sensing performance considering both bulk and molecular surface
sensitivities, highlighted the platform's effectiveness. These results position the AuNR-coated
glass substrates within the advancements of the broader nanoplasmonic biosensing field.
Plasmonic Characterization
Transmission-mode UV–vis spectroscopy experiments were conducted to characterize the
plasmonic properties of short and long gold nanorods (AuNRs) in bulk solution and the
deposited state. The optical extinction spectra revealed localized surface Plasmon resonance
(LSPR) behavior with distinct peaks corresponding to transverse and longitudinal Plasmon
modes. The focus was on the experimentally determined longitudinal peak's λmax position,
which varied based on the local dielectric environment either in solution or deposited on a glass
substrate.
For short AuNRs, the longitudinal λmax shifted from ~665 nm in bulk solution to ~680 nm when
deposited, aligning with theoretical calculations. Theoretical analyses modeled the AuNR as a
prolate spheroid with spherical cylinder geometry, considering the wavelength dependent
dielectric function of gold. The calculated longitudinal λmax positions were ~683 nm and ~710
nm in bulk solution and the deposited state, respectively. Similarly, long AuNRs exhibited a shift
from ~814 nm to ~831 nm experimentally, in agreement with theoretical calculations of ~780 nm
and ~820 nm in bulk solution and the deposited state.
These findings indicate that the longitudinal λmax position of AuNRs is sensitive to changes in
the local dielectric environment during colloidal deposition. The substantial contact area between
AuNRs and the glass substrate significantly influences plasmonic properties, distinguishing it
from the negligible shifts observed in the colloidal deposition of other nanostructures like
spherical gold nanoparticles.
Bulk Sensitivity Measurements
The bulk refractive index sensitivities of short and long gold nanorods (AuNRs) were
experimentally characterized in both bulk solution and the deposited state by measuring
longitudinal λmax shifts in water-glycerol mixtures (0–35% v/v glycerol). The longitudinal λmax
position increased with higher glycerol fractions, demonstrating a responsive behavior to
changes in bulk refractive index within the tested range.
For short AuNRs, linear trends were observed in plots of λmax shift against the change in bulk
refractive index units (ΔRIU), yielding sensitivities of 277.5 nm/RIU in bulk solution and 167.3
nm/RIU in the deposited state. The diminished sensitivity for deposited short AuNRs is
attributed to their substantial contact area with the glass substrate. Similarly, long AuNRs
exhibited linear responses with sensitivities of 419.5 nm/RIU in bulk solution and 236.6 nm/RIU
in the deposited state, comparable to Au nanodisks on a glass substrate. Analytical calculations
supported the experimental results confirming the ~40% decrease in sensitivity for deposited
AuNRs due to substrate contact.
The bulk refractive index sensitivities of short and long AuNRs surpassed those of suspended
and deposited spherical Au nanoparticles, emphasizing the high sensitivity of AuNR-coated glass
substrates. These findings motivated further investigation into surface sensitivities in response to
biosensing events near the sensor surface.
Surface Sensitivity Evaluation
In liquid-phase biosensing experiments using AuNR-coated glass substrates within a
microfluidic chamber, real-time adsorption of 100 μM bovine serum albumin (BSA) protein
revealed rapid increases in the λmax signals, reaching saturation with around 6 nm and 16 nm
shifts for short and long AuNRs, respectively. Conversion of λmax shifts to refractive index units
(RIU) demonstrated values of 0.04 and 0.06 RIU at saturation for short and long AuNRs.
Subsequent investigation of biosensing capabilities involved detecting the transformation of
adsorbed vesicles into supported lipid bilayers (SLB) using an amphipathic α-helical (AH)
peptide, showcasing significantly larger responses for long AuNRs compared to short AuNRs,
indicating over two-fold improved sensing performance.
Biosensing Performance Comparison
In nanoplasmonic biosensors, the widely used metric, bulk refractive index sensitivity, is vital for
evaluating sensing performance, emphasizing the importance of both surface and bulk
sensitivities in biosensing measurements. The comparison of normalized refractive index unit
(RIU) shifts for BSA adsorption against bulk refractive index sensitivity across various Au
nanoplasmonic platforms reveals that surface and bulk sensitivities are distinct metrics. The
AuNR-coated glass substrates, especially the long AuNR array, demonstrate remarkable surface
sensitivity, surpassing responses from traditional SPR platforms. This improvement is attributed
to significant electromagnetic field enhancement around high-aspect-ratio Nanos-structures,
tightly confined field enhancement, and the absence of a surfactant layer on the AuNR surface.
The ensemble averaged readout in measurements allows for collective representation of
individual AuNR signals, offering a notable advantage for potential single-molecule detection, a
capability not dependent on array arrangement, distinguishing it from other plasmonic sensing
modes.

Conclusion
Researchers have been exploring different shapes and sizes of nanostructures to improve the
performance of biosensors. They are increasingly focusing on improving the surface sensitivity
of nanoplasmonic biosensors, which detect tiny amounts of biological molecules. While most
previous research used simple shapes like circles, this study shows that long, thin Nano rods
(called "Nano rods") are even better for this purpose. These Nano rods capture and concentrate
light more effectively, making them more sensitive to changes on their surfaces, like when a
protein molecule binds to them. This is important for building better biosensors that can detect
diseases and other health problems earlier and more accurately. The study also emphasizes that
focusing on surface sensitivity, rather than just how much light the sensor absorbs, is crucial for
designing high-performance biosensors. They found that using laterally anisotropic
nanostructures, such as Nano rods, can lead to better results than using laterally isotropic
nanostructures, such as circular Nano disks. This is because the anisotropic nanostructures have a
higher aspect ratio, which leads to larger field enhancement effects and shorter decay lengths.
These factors contribute to the high surface sensitivity of the sensors, which is important for bio
sensing. The researchers also emphasize that surface sensitivity is a more critical performance
metric than bulk sensitivity for designing high-performance nanoplasmonic biosensors.

Figure 1 Overview of experimental strategy. Top row: Sensor fabrication involved the colloidal deposition of short and long
AuNRs with distinct length (L), diameter (D), and aspect ratio (A.R.) values. Photographs of the aqueous AuNR dispersions and
AuNR-coated glass substrates along with scanning electron microscopy (SEM) images of the AuNR-coated glass substrates.
Middle row: Plasmonic characterization of short and long AuNRs in the bulk solution and in the deposited state based on
experimental measurements and analytical calculations. Bottom row: Schematic illustration of protein adsorption and vesicle-
peptide interaction experiments to evaluate real-time mea- segment performance of the AuNR-coated glass substrates with
respect to surface sensitivity, as well as biosensing performance comparison with other nanoplasmonic sensing platforms.
Figure 2 Plasmonic characterization of solution-phase and deposited AuNRs. Optical extinction spectra of (a) short (low aspect
ratio) and (b) long (high aspect ratio) AuNRs in bulk solution and deposited on a glass substrate. Comparison between
experimentally determined and analytically calculated longitudinal λmax positions for (c) short and (d) long AuNRs in bulk
solution and in the deposited state. All measurements were conducted in water. Error bars represent standard deviation from n = 3
measurements
Figure 3 Bulk refractive index sensitivities of solution-phase and deposited AuNRs. Optical extinction spectra of deposited (a)
short and (b) long AuNRs in the presence of different water/glycerol mixtures (0–35% v/v glycerol). Experimentally measured
bulk refractive index sensitivities of solution-phase and deposited (c) short and (d) long AuNRs based on the longitudinal _λmax
shift responses in water/glycerol mixtures with different refractive index values. The lines are linear best-fits of the measurement
data and the reported bulk refractive index sensitivity values are reported from the corresponding slopes. Comparison between
experimentally determined and analytically calculated bulk refractive index sensitivities of (e) short and (f) long AuNRs in bulk
solution and in the deposited state. Data are reported as mean ±standard deviation from n = 3 measurements