Doctoral Research Summary

Doctoral Research Summary

I do multi-disciplinary research to integrate the fundamental concepts of functional proteomics, microfluidics, and nanofabrication. I employ principles of theoretical modeling and simulations, microfluidics, surface chemistry, and nanophotonics for the realization of a novel integrated lab-on-chip (LOC) platforms for pre-concentration, filtration, and label-free detection of proteins and biomarkers. In my doctoral research, I primarily focused on investigating and demonstrating a compact LOC sensor for the post translational modification (PTM) profiling of the proteins used as biomarkers (i.e., PSA), for early prostate cancer detection. The LOC sensor comprised of an on-chip pre-concentration and filtering device to isolate the targeted low-abundance proteins form the complex input biological samples and an integrated photonic sensor microarray for profiling the molecular structures of the isolated protein epitopes. The motivation of this work has been extracted from the inability of conventional affinity-based pre-concentration and separation techniques for elucidating PTMs of the proteins, and the unavailability of LOC sensors for this task.

Doctoral Research Overview

Demonstration of affinity-based multiplexed biomolecular capturing and release using miniaturized Polydimethylsiloxane (PDMS) micropillar-based platform

Towards the goal of "early disease detection", and particularly cancer detection, monitoring the concentration of biomarkers (proteins) in a biological sample is of utmost importance. A typical sample of blood contains more than 10,000 different proteins with different orders of varying concentrations [1]. The analysis for a specific protein in such a high background noise results in decreased sensitivity, dynamic range, specificity and efficiency. Moreover, the proteins are extremely sensitive to the ambient environment including temperature, pressure, humidity, pH, ionic strength, etc. [2]. This makes the detection extremely cumbersome in low concentration and low quantity samples. The sensitivity, diversity, and complexity of the biological samples, along with the need to specifically determine an analyte (protein), have forced the researchers to explore and develop efficient biomolecular pre-treatment (separation, purification) and detection technologies. Various sample pre-treatment functions such as cleaning, filtration, mixing, heating, and concentration before the analysis, have been demonstrated on-chip [3], which are traditionally performed in on-chip environment.

Pre-concentration improves sensitivity of detection and reliability of analysis by significantly improving signal-to-noise ratio (SNR), especially when high performance optical detection is employed. Extremely small sample volumes are required of the order of nanoliters and picolitres, compared to milliliters of volumes required for routine on-chip sample analysis, resulting in reduced consumption of the expensive biological samples and reagents [4].

Surface binding techniques, in particular solid phase extraction (SPE), immunoprecipitation [5], and affinity based separation methods have found much interest of the researchers for protein enrichment [4]. However, harsh conditions, such as denaturing buffer [6], acid cleavage [7], or treatment with excess biotin [8], are needed for the release of the surface captured proteins (i.e., breaking of the affinity bond is required). These methods can result in sample degradation, introduction of contaminants (due to the release of non-specifically bound proteins), elution of endogenous proteins (along with the desired protein), etc. [6].

Addressing the above mentioned challenges, I have demonstrated biomolecular (i.e., proteins and biomarkers) filtering and pre-concentration based on an affinity-based separation approach. The target biomolecules are captured and pre-concentrated on the PDMS-based micropillars. The micropillar's surface is specifically functionalized with the specific affinity epitopes corresponding to the biomolecules for enhanced affinity-based capturing. The pre-concentrated biomolecules are then released on demand and can be recaptured on a sensing transducer (e.g., optical sensor arrays), allowing a complete label-free sensing and profiling (secondary epitopes such as glycoforms) of the biomolecules [9].

I employed a multi-step surface functionalization protocol to optimize on-the-ow surface functionalization and capturing efficiency of the biomolecules in a PDMS-based microfluidic platform. I designed, optimized, fabricated, and characterized a PDMS-based microfluidic platform, having a micropillar based biomolecular pre-concentration chamber allowing increased surface-to-volume ratio (SVR) for enhanced biomolecular filtering and pre-concentration. The surface of the pre-concentrator chamber is functionalized with the Avidin protein, followed by the use of a biotinylated photo-cleavable (PC) linker conjugated with the biomolecular specific antibodies (i.e., anti-PSA IgGs). The use of PC-biotin linker is the key towards solving the challenges such as sample degradation, introduction of contaminants (due to the release of non-specifically bound proteins), and elution of endogenous proteins (along with the desired protein) [6], faced while releasing the pre-concentration samples from microfluidic devices [9].

I effectively demonstrated the capture and release of IgG proteins (from Rabbit serum) and PSA cancer biomarkers (fPSA, human serum) using the PC-biotin linker with clinically relevant sample concentrations and volumes. I also demonstrated the capability of multiplexed glycoprofiling for elucidating the post translational modifications (PTMs) of the PSA cancer biomarker using the Sambucus Nigra Lectin (SNA) and Maackia Amurensis Lectin II (MAA-II) lectins. A dynamic working range of 10 orders of magnitude higher compared the commercially available ELISA kit for PSA detection (i.e., 0.01 ng/ml to 12 ng/ml for fPSA ELISA kit [10]), has been achieved using the designed PDMS-based micropillar pre-concentration platform [9].

Demonstration of nanoporous MWCNT forests and micropillars for multiplexed on-chip biomolecular capturing and release

Apart from the role of the PDMS material, the LOC biomolecular pre-concentration and filtration has greatly benefited from the development of nanoporous materials. Efficient biomolecular separation using nanoporous materials requires controllable pore sizes, length and surface chemistry [11]. The pores are required to have a uniform diameter distribution in order to achieve highly selective filtration. Also, high porosity is required to obtain desired analyte flux [12]. Porous designs allow fluid flow both around and through the porous elements, enhancing physical interactions between the particles in the flow and the functionalized porous surfaces [13]. In addition, the large SVR of the porous materials increases the functional surface area in a given volume and presents an enhanced opportunity to target and isolate multiple particle types using a single device [13]. Furthermore, the integration of nanoporous materials in microfluidics enables access to a largely unexplored spectrum of smaller sub-micrometer species that the current LOC bio-MEMS platforms are trying to interact [14].

I demonstrated the use of carbon nanotubes (CNTs) as high porosity material for enhanced on-chip biomolecular pre-concentration applications. I fabricated and utilized CNTs forests and micropillar structures over the silicon substrate to increase the effective surface area of a microfluidic pre-concentrator device. These CNTs grown are multi-walled (concentric walls) with an average height of 30 um, average diameter of 8 nm to 12 nm, and an average intra-CNT spacing of 80 nm to 100 nm, leading to a volume fraction of less than 1% to 2% CNTs. This resulted in an increased surface are for biomolecular capturing applications [9].

The MWCNT forests and micropillars has been grown over the silicon substrate, aligned, and bonded with the PDMS based microfluidic channels to develop the biomolecular preconcentration and filtering devices. The capturing efficiency and performance of these designed MWCNT forests and micropillar-based pre-concentration platforms have been assessed by employing the PC-biotin linker-based modified lectin immunoassay for the PSA biomarker capturing, pre-concentration, glycoproling, and release. The capability of multiplexed glycoproling for elucidating the PTMs of the PSA cancer biomarker has been demonstrated by using the SNA and MAA-II lectins [9].

Demonstration of high-Q SOI microresonators with high repeatability, for multiplexed and array based sensing applications

The on-chip silicon optical microring resonators are planar in configuration, operate in single radial mode (TE or TM) with high Q-factors, and allow high SNRs. This allows realization of extremely dense and highly multiplexed sensor platforms with ultra-ne spectral resolutions. Moreover, the ease of fabrication of planar devices in silicon and CMOS process compatibility not only enables complete System-on-a-Package (SOP) realization for biosensing applications, but also allows much quicker lab-to-market transition of these devices. The challenge lies in the small sensing area of these resonators for analyte adsorption compared to the overall footprint. This results in small optical field overlap between the microresonator and the analyte [15], thereby resulting a in Poisson-limited sensitivity for detection of larger biomolecules [16]. Moreover, the temperature induced drifts and variations in silicon microresonators, if not properly addressed, result in inaccurate measurements [17].

I have designed high-Q optical microring and spiral resonators in SOI platform to work as highly sensitive, compact, and scalable transducers, enabling a versatile realization of highly multiplexed and array based chemical and biological sensing platforms. I have demonstrated the integration of microfluidics with the sensing transducers, allowing on demand delivery of extremely small volumes of chemical and biological analytes, resulting in realization of advanced LOC solutions for various chemical and biological sensing applications [9].

A highly multiplexed, high-Q microring structures has been demonstrated which allows independent and individual monitoring of each of the microring sensing element at its corresponding wavelength. The platform has been design in such a fashion that when simultaneously excited, the corresponding resonance frequencies are evenly spaced in the desired FSR range without any spectral overlap [9]. Similarly, a high-Q spiral resonator based sensing platform has been demonstrated, providing a higher sensing area in a small footprint. The designed spiral resonator platform provided a small footprint of 50um by 50um, and a sensing area more than times of a microring in a similar footprint. Q-factors of 50K has been achieved for both of the designs. The sensitivity performance of ~0.43 pg/(mm2/pm), a minimum limit of detection (LOD) for mass sensing as ~0.5 pg/mm2, and a minimum resolution of ~1 pm has been demonstrated for biosensing applications [18].

Another challenge in silicon nanophotonics lies in lighting up these microresonator devices (i.e., coupling light in these integrated on-chip optical platforms) to generate enhanced light-matter interaction for advanced biosensing applications. On-chip grating couplers are realized for this purpose which allow relaxed fiber-coupler alignment, decreased CMOS real-estate, polarization dependent operation, and ease of fabrication [19],[20]. Key challenges in the grating coupler design includes back reflection, fiber-coupler mode matching, directionality, and coupler coupling efficiency. Several designs of high efficiency grating couplers have been presented in the literature to address these issues. These include fabrication of apodized grating [21], sub-wavelength gratings [22], partially etched grating [23], and anti-reflection mirrors [24]. These modifications result in exhaustive simulations and design optimizations, increased fabrication complexity, and overall cost of the devices. Moreover, the literature is more focused towards design of TE polarized input/output (I/O) grating couplers for high performance communication and optical computing applications where coupling efficiency is an important aspect, while less attention has been given to TM polarized I/O grating (highly evanescent nature of TM polarized light results in higher losses) [25]. In the realm of biosensing applications, the aspect of coupling efficiency is relaxed compared to the high performance on-chip optical communication and computing applications [26],[27]. However, the overall cost of the device is of key importance for the realization of disposable point-of-care (POC) on-chip optical biosensing platforms.

I designed, optimized, fabricated, and characterized fully etched TM-mode grating coupler with an efficiency of 50%, using a duty cycle (D) of 0.555, and a grating pitch of 0.996 um. Actual TM polarized grating couplers are then fabricated along the microresonators to demonstrate the CMOS compatible fabrication of densely integrated, grating based optical sensing platforms.

Another aspect of my design approach is the optimization of the nanofabrication processes, and repeatability of fabrication for these sensing platforms. I have designed and demonstrated these devices in a manner to be compatible with single step electron beam (e-beam) lithography, single step etching, and extremely low-cost industrial grade microfluidic platform integration. This has enabled me to achieve lower cost of fabrication, device complexity, and much faster lab-to-market time [9].

References

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