An optical label-free aptasensor based on dye doped leaky waveguide (DDLW) for biomarker detection

Alamrani, Nasser A.

Abstract

Due to the rapid increase in routine measurement of biomarkers, such as a protein, in blood samples for healthcare monitoring, different techniques have been developed to facilitate this measurement in terms of cost-effective and fast measurement. Label-free optical biosensors are an attractive technology because they do not require fluorescent dyes or radioactive isotopes for detection, thereby reducing cost and measurement time.
Dye-doped leaky waveguides (DDLW) were developed by our group in 2016 as a label-free and a real-time optical biosensor measurement. They measure the change in the refractive index in a real-time when an analyte interacts with the waveguide. DDLW is a leaky optical mode which features a lower index waveguide material such as a hydrogel. This is advantageous for the sensitivity of the sensors as the analyte can be inserted into the sensing region and hence interact with a large portion of the confinement light, resulting in great enhancement of the sensor signal. Furthermore, DDLW shows other advantages such as low cost and easy fabrication.
In this work, a DDLW was developed as a label-free and a real-time optical biosensor measurement with using for the first time a chitosan hydrogel polymer as a porous waveguide layer. The inexpensive hydrogel offers advantages such as non-toxicity and biocompatibility and also features functional amino groups that are amenable to tethering of bio-recognition elements. Furthermore, aptamers were chosen as the bio-recognition element to capture the analyte due to their selectivity, stability and low cost compared to more commonly used antibodies. This was the first time the possibility of using an aptamer as a bio-receptor in the 3D network leaky waveguide containing chitosan porous hydrogel on glass substrate.
Thrombin and prostate specific antigen (PSA) were selected as key biomarkers for measurement by the fabricated DDLW device. Thrombin is an allosteric serine protease that works as the central protease in the coagulation cascade. With some critical role in the coagulation process, thrombin is linked to Parkinson’s and Alzheimer’s diseases. Prostate specific antigen has been considered the most validated biomarker in serum for early detection of prostate and breast cancers.
The preliminary result was that chitosan could be successfully prepared to provide a single waveguide mode by using 2% of chitosan solution coated at speed of 3000 rpm. The waveguide obtained showed a high sensitivity to the value of refractive index.
However, the porosity of the waveguide was found to be small, which prevented diffusion of the large molecules into the sensing region. Different methodologies were utilised to enhance the waveguide’s porosity. Using a lower concentration of chitosan with a controlled drying time for the coated wet chitosan film was found to be effective at improving the pore size of the waveguide. The best conditions was found to be a 1% chitosan solution coated at spin speed of 900 rpm with 3 min of drying time of the coated film.
For the detection of thrombin, an aptamer molecule was immobilised into the porous chitosan waveguide via a streptavidin and biotin complex. First, the attachment of streptavidin was optimised using three methods: covalent attachment, non-covalent attachment and physical adsorption. Comparing the protocols uses, covalent attachment using glutaraldehyde as the cross-linker exhibited the highest amount of streptavidin immobilised onto the waveguide. An anti-thrombin biotinylated aptamer was then attached to the immobilised streptavidin. The detection of thrombin was achieved by observing a rapid shift in the resonance angle upon applying 1μM of thrombin solution for 15 min. The sensitivity of the sensor to detect thrombin was found to be enhanced upon increasing the incubation time of thrombin molecules. Based on this, a calibration curve was then obtained for different concentrations of thrombin, and the limit of detection (LOD) of the sensor was found to be ≈3.7 nM after one hour of incubation. This limit of detection (LOD) was comparable to those were previously reported for detection of thrombin such as SPR. The applicability of the DDLW device for the measurement of thrombin in a clinically relevant sample was evaluated. It was found that the shift observed for the first four hours of incubation of spiked thrombin (50 nM) in 10 % human serum sample was close to the shift obtained when 50 nM of thrombin was measured in an aqueous buffer. Therefore, it was concluded that thrombin was successfully detected in a diluted serum sample.
The detection of PSA was more challenging as compared to the detection of thrombin. An enhancement was obtained upon the thermally treated anti-PSA aptamer prior to immobilisation. This was thought to prevent the folding of aptamer molecules and hence improve the binding of PSA. The detection of PSA was comparable to the detection of thrombin with the thermal treatment of aptamer. Based on this, a series of concentrations of the PSA ranging from 25 nM and 75 nM were then successfully detected in an aqueous buffer. It was concluded that the fabricated DDLW device could be applied to other applications using an aptamer as a recognition element.

Citation

Alamrani, N. A. An optical label-free aptasensor based on dye doped leaky waveguide (DDLW) for biomarker detection. (Thesis). University of Hull. https://hull-repository.worktribe.com/output/4749483

• Thesis Type: Thesis
• Deposit Date: Jul 31, 2024
• Publicly Available Date: Jul 31, 2024
• Keywords: Chemistry
• Public URL: https://hull-repository.worktribe.com/output/4749483
• Additional Information: Department of Chemistry; University of Hull
• Award Date: Jul 1, 2019