Lab 11 Nanophotonics Introduction As we have seen throughout the course microscopy and spectroscopy are powerful tools in biological sciences In recent years advances in nanotechnology have also improved microscopy and spectroscopy by increasing their sensitivity through coupling with nanostructures Biomedically relevant molecules such as antigens or antibodies in low concentrations can be detected using spectroscopy with the help of nanotechnology as we will see in this lab Part 1 Gold nanohole array AuNHA for biosensing Gold is a reflective material however if patterned with a repeating hole pattern it can have enhanced transmission even when the holes are smaller than the wavelength of the light Fig 1 Fabricated AuNHA for biosensing experiments This effect is called extraordinary optical transmission EOT and it is enabled by surface plasmon resonance SPR The explanation of both effects is beyond the scope of this course but SPR is very sensitive to refractive index changes on the gold surface This in turn changes the spectral response of the AuNHA Different molecules have different refractive indices and therefore we can deduce the presence of biomolecules on the AuNHA surface through the change in spectral response Research groups have functionalized the conjugate of target biomolecules on the AuNHA surface to detect the presence of biomarkers within a solution illustrated in Fig 1 For example in a recent work from Yesilkoy lab SARS CoV2 proteins were functionalized on AuNHA to detect the presence of anti COVID 19 antibodies illustrated in fig 2 Fig 2 Detecting biomolecule through the change in refractive index on the surface The spectra will shift towards a longer wavelength red shift From Adi W et al Biomed Opt Express 13 2130 2143 2022 For this lab two AuNHAs one uncoated and another one coated with photoresist on the order of several microns have been prepared for you This photoresist layer is supposed to simulate a layer of biomolecule Depending on the sequence protein size varies from nm to tens of nm Compared to this microns thick material is rather thick and represents a bulk quantity of sample but this will suffice to represent a biological measurement Do illumination 1 Prepare your Kohler illumination and close down the aperture stop coherent 2 Focus on the surface of uncoated AuNHA take an image with appropriate exposure 3 Take off your objective and replace it with the spectrometer 4 Take spectra of the uncoated AuNHA Focus on the wavelength region between 400 and 5 Repeat steps 1 4 with the coated AuNHA Change the settings accordingly to obtain 1000nm your signal will be located there similar spectra and images for both AuNHA 6 Take the spectra of your light source You might have to change your settings to obtain peak heights similar to the data from your AuNHAs Across all the data that you take you have to ensure that the signals are about the same height For more sophisticated experiments the data must be normalized properly by taking the illumination signal into account similar to what you did in lab 10 However in AuNHA measurement is slightly more complex and we will need only the peak positions of the spectra Thus normalization can be omitted if it is distorting the spectra too much The coated peak will redshift and you will calculate the shift in nm as a function of refractive index change in refractive index unit for your lab report The bulk refractive index n of photoresist is about 1 5 similar to protein about 1 4 However at small thickness similar or close to the wavelength of light the effective refractive index neff also depends on the thickness of the material Later in the lab report you will calculate how much the peak shifts as a function of thickness Note The Fabry Perot effect similar to the ones we saw in lab 10 can also be seen here but more pronounced thanks to the enhanced light matter interaction provided by the AuNHA Similar to lab 10 you will deduce the thickness of the photoresist and use this value to check the sensitivity of your AuNHA Part 2 Nano plasmonic effect and gold nanoparticles Some nanotechnologies are not just based on devices fabricated using photolithography but also through nanoparticle synthesis A scanning electron microscopy SEM image is shown in fig 4 Synthesized nanoparticles can be used in wide variety of biomedical applications such as for drug delivery photodynamic therapy labeling agent and many more More information about them can be found here Gold Nanoparticles Properties and Applications sigmaaldrich com Fig 4 SEM images of gold nanoparticles From Gold Nanoparticles Properties and Applications sigmaaldrich com Gold nanoparticles of different sizes have different optical properties This is illustrated in fig 5 Fig 5 Visual illustration of gold nanoparticles of different sizes top and spectra of some sizes bottom For this lab we will take the spectra of various nanoparticle sizes 5nm 50nm 80nm 250nm Your instructors will hand you 4 bottles of different nanoparticle sizes you will not know which ones are which initially Do 1 Still with your setup from part 1 take off the objective and replace with spectrometer 2 Open your aperture stop and adjust your integration time accordingly 3 Similar to lab 10 get the spectra of your illumination light on trace A 4 Take spectra of all bottle on trace B one by one and divide to obtain your real particle spectra on trace C exactly like in lab 10 Focus on the wavelength region between 400 to 1000nm your signal will be located there Save all spectra as images and data points to plot later Wrap up Clean your lab space as usual Lab write up Part 1 1 Plot the spectra from your both coated and uncoated AuNHAs and the light source in one window Bonus point if you can select plot in the wavelength range of 550 to 1000nm 2 Calculate the peak wavelength shift between coated and uncoated peaks as shown in fig 3 3 Estimate the thickness of the photoresist using the Fabry Perot peaks using the equation for the gap measurement between two glass slides from lab 10 4 Use this value to calculate the wavelength shift per sample thickness of your AuNHA This characterize the sensitivity of your device 5 Now imagine you have 300nm of protein samples instead what peak shift would you expect Comment on the values AuNHA is far more sensitive on its surface than far from it so the values that you got will probably be far from accurate but this will give you an idea about how the sensor is used Part