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Multiplexed Biosensors for Pathogen Detection

  • Author(s): Gopalakrishnan Meena, Gopikrishnan
  • Advisor(s): Schmidt, Holger
  • et al.

Efficiently combating infectious diseases is a vital part of primary health care. The first step taken towards treating diseases is detection and identification. Disease detection has evolved to become an essential unit in both hospitals, clinical labs and now even at the point of care, to help physicians make well informed diagnosis and treatment in a timely manner. For quick response to an infection, all of these health care sectors need diagnostic tools which are sensitive enough to detect pathogens at an early stage. Integrating them with sample preparation units expedites the diagnostics process through automation, removing human errors and minimal use of clinical samples. Adding multiplexed detection capabilities to these systems makes diagnosis faster through parallel processing. It also helps obtain more information of the disease by simultaneously probing for different characteristics of the pathogen such as the type of species, mutations or even cross reactivity with other targets. Optofluidics has emerged as one of the most desired technology to incorporate all of this. This thesis focuses on several multiplexed biosensing techniques using optofluidic biosensor chips that have planar solid core (SC) and liquid core (LC) waveguides and their combination with integrated sample preparation units. Platforms using both silicon based anti-resonant-reflecting-optical-waveguide (ARROW) systems and polydimethylsiloxane (PDMS) based optical waveguide systems are described.

Multiplexed detection is executed in the ARROW biosensor platform using multi-mode-interference (MMI) waveguides which create wavelength dependent excitation patterns in the liquid core ARROW enabling fluorescence based multiplexed detection of biomolecules. The thesis describes how the signal-to-noise ratio of the ARROW biosensors is improved more than 30x using a buried oxide layer and how the design of the MMI waveguides is optimized for the buried waveguides to generate better excitation profiles for the desired spectrum, extending from 488 nm to 738 nm. The improved ARROW platform is used to demonstrate 7x multiplexed detection of an antibiotic resistant bacterial panel by combining spectral multiplexing with a three-color combinatorial labeling scheme. The ARROW biosensor platform is also used to demonstrate for the first time, simultaneous amplification-free detection of three antibiotic resistant nucleic acid biomarkers (bacterial plasmids) with single molecule sensitivity using spatial multiplexing. Finally, pertaining to the COVID-19 pandemic which started in the year 2020, spectral and spatial multiplexing techniques are combined to demonstrate the first dual detection of SARS-CoV-2 RNA and protein targets, both with single molecule sensitivity. Also presented is a novel SiO2 based biosensor with index guided SC and LC waveguides. The optical mode of the LC waveguide is guided and tuned by using high refractive index ZnI2 salt solution for greatly enhanced fluorescence detection. This enables sensing nanobeads with an ultra-low limit of detection down to clinically relevant attomolar range. The thesis concludes with a report on a PDMS based sensor integrating monolithically both a sample preparation unit and sensing system. The device has pneumatically activated microvalves, which is used to capture, filter and tag Zika virus specific nucleic acid and protein targets. These are detected simultaneously in the same chip using PDMS based optical waveguides for differential detection of ZIKA virus from other cross reacting species such as Dengue.

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