Conclusiones

A further step in the fabrication of the immunosensor has been the study of a method to transfer it from a rigid substrate (Si in this case) to a polymeric flexible one, with the aim to improve the integrability and biocompatibility for

CHAPTER 4 – PROOF OF CONCEPT OF A LABEL-FREE IMMUNOSENSOR AND FURTHER FABRICATION STEPS

in-vivo assays.

The steps of the fabrication protocol are the following (Figure 4.12): a layer of AR-P 6200 resist has been spun on the layer of Si3N4 with a spin-speed of 2000 rpm for 60 s and post baked at a temperature of 180°C for 10 minutes. The resist has been patterned with an EBL process with voltage of 50 kV and area dose of 130 μC/cm2 _{and developed in xylene for 2 minutes. The transfer of the PhC} patterning on the Si3N4 layer has been achieved by RIE using the following recipe:

 CHF3: 58 sccm

 O2 : 2 sccm

 Pressure: 93 mTorr

 Potenza: 360 W

 Preconditioning time: 3 minutes

 Time: 17 minutes

Finally the membrane has been release using a dry etching in a solution of deionized water with the 3% of TMAH concentration for 15 minutes at 100°C. In Figure 4.13 is reported a picture of the 1 mm × 1 mm PhC membrane acquired by optical microscope. The darker colour in the centre of the membrane is caused by the absence of Si underneath and by means of this colour variation it is possible to check the progress of the wet etching (Figure 4.12 step 3) during the membrane releasing. Finally, the two lines passing in the middle of the structure are due to the stitching error, that is the mismatching between the write-fields during the EBL process.

CHAPTER 4 – PROOF OF CONCEPT OF A LABEL-FREE IMMUNOSENSOR AND FURTHER FABRICATION STEPS

Figure 4.12: Fabrication process for the transferring of the PhC membrane on flexible substrate.

Figure 4.13: Image acquired by optical microscope of the PhC 1 mm × 1 mm membrane after the wet etching in TMAH solution. The colour variation in the centre of the structure represents the absence of Si underneath while the lines passing in the middle are caused by the EBL stitching error.

Then the sensor has been transferred on a thin layer of polydimethylsiloxane (PDMS n 1.4) using the process described by M.G. Scullion et al.[46]. The PDMS has been prepared by mixing 5 ml of silicon elastomer with 0.5 ml of curing agent and degassed. After that, a thin layer (less than 1 mm) has been deposited in a plastic box and has been left in oven at 60°C for several hours. At this point, the sample has been pressed on the PDMS and then peeled off, leaving the membrane attached on the polymer (Figure 4.14). The transferred structure has been measured revealing a peak of reflectivity

CHAPTER 4 – PROOF OF CONCEPT OF A LABEL-FREE IMMUNOSENSOR AND FURTHER FABRICATION STEPS

Figure 4.14: Picture of an experimental test about the transferring of PhC structures on PDMS. The sample is placed on a microscope coverslip. The little fragments between the larger structures are pieces of membranes detached during the TMAH etching (Figure 4.19 step 3). The complete membrane transferred has an area of 1 mm × 1 mm. The process needs further optimizations to increase the number of complete transferred structures.

Figure 4.15: Reflectivity spectrum of the PhC (p = 460 nm r = 0.25*p) on PDMS measured in air.

A further step of this activity will be the design of a new PhC structure to be transferred on PDMS, optimizing its electromagnetic properties for biosensing applications and opening the way to a new generation of nanofabricated devices characterized by high resolution, integration and biocompatibility.

CHAPTER 4 – PROOF OF CONCEPT OF A LABEL-FREE IMMUNOSENSOR AND FURTHER FABRICATION STEPS

4.4 Summary

In this chapter has been presented the proof-of-concept of a label-free immunosensor for the revelation of biomarkers in solution. Initially, the reflectivity spectrum of the sensor has been tested in different refractive index solutions, showing a diverse shift of the reflection peak wavelength in each one. Subsequently, it has been functionalized with a layer of antibodies for the revelation of the IL-6 biomarker, building the calibration curve and determining a limit of detection of 1.5 pg/ml, quite below the concentration of the protein in a ill patient.

In the last part of the chapter the activity carried out at the Photonics Group of the University of York has been discussed. It has been studied the fabrication process to achieve completely free standing membrane for an innovative procedure for the transferring of PhC biosensors on a tip of an optical fiber to realize biosensors for in-vivo detection of proteins concentration. Finally, some experimental tests are illustrated about the optimization of the process for the transferring of PhC membrane sensors on a polymeric flexible material.

Conclusion

The aim of this thesis has been the design, fabrication and testing of a label-free immunosensor for the revelation of biomarkers in solution. The project has been a collaboration between the IIT-CBN centre, Politecnico di Bari and the Mawson Institute (University of South Australia).

To get the goal, a 2-D photonic crystal (PhC) slab with square lattice has been designed by means of numerical simulation using the finite difference time domain (FDTD) method. The structure consists in a 1 mm × 1 mm area Si3N4 with thickness of 300 nm. The geometrical parameters like lattice period and radius has been optimized getting reflectivity peaks around 640 nm and 760 nm.

The designed structure has been fabricated by means of a nano-fabrication protocol composed by e-beam lithography, dry and wet etching. Each step has been optimized achieving high quality membranes with high reproducibility as it has been proved by morphological characterization by means of scanning electron microscopy.

The PhC membrane has been functionalized by means of a layer of antibodies for the revelation of the interleukin-6 protein, a biomarker in relation with diseases like oral cancer and Alzheimer. The immunosensor has been tested in the revelation of the protein in solution, building a calibration curve. The detection limit determined from the tests has been of 1.5 pg/ml, a lower quantity than the usual concentration of the protein in ill patients.

In the last part of the activities (carried out at the Photonics Group of the University of York (UK)) further optimizations of the fabrication protocol of PhC membranes for biosensing applications have been proposed. In particular, a complete free standing PhC membrane with area 300 µm × 300 µm has been obtained, which will be used in the process for the transferring of biosensors on a tip of an optical fiber to realize biosensors for in-vivo detection of proteins concentration.

membranes from rigid substrate to polymeric layers have been performed, procedure that will be optimized for the realization of high resolution and biocompatibility biosensors.

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