Development of a concentration-enhanced mobility shift assay platform for aptamer-based biomarker detection and kinase profiling

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2012.Cataloged from PDF version of thesis.Includes bibliographical references.New methods to quantify rare biomarkers from patient samples are critical for developing point-of- care diagnostic platforms. To be compatible with resource limited settings, these assays have to provide fast and accurate results without sacrificing ease of use. Biosensing in homogeneous fashion is the preferred format which satisfies these criteria, but the lack of amplification method is a bottleneck that limits their use for sensitive applications. To address this issue, this thesis explores physical signal amplification means to increase the sensitivities of homogeneous assays. We identified several key applications where the use of these technologies could make a positive impact in improving medical diagnostics systems and advancing biological research. We first outline the use of electrokinetic concentration to realize a continuous signal amplification scheme that increases the sensitivity of homogeneous mobility shift assays. By simultaneously concentrating and separating reacted and unreacted species (with different mobilities) in this device, we can perform sensitive, quantitative and ratiometric measurement of target biomarkers. Using this platform, we improved the sensitivity of aptamer affinity probe capillary electrophoresis to achieve pM detection limit of IgE and HIV-RT in simple buffer and serum sample. This work is timely and impactful as it directly addresses the sensitivity shortcomings of using aptamers as low cost and robust substitutes for antibodies in point-of-care applications. Next, we presented a herringbone nanofilter array device which can perform continuous sizes-elective concentration of biomolecules based on their direct interaction with nanostructures with comparable critical dimensions. We demonstrated the use of this platform to perform a novel homogeneous immunoassay for detecting a cardiac biomarker, C-reactive protein, at clinically relevant concentrations. Finally, we demonstrated that the concentration-enhanced mobility shift assay platform is a powerful tool for probing biological activities such as cellular kinase activities. We have developed technology to isolate, grow and lyse single cells, and used our platform to measure kinase activities from single cells. Through rational design of peptide substrates and spacers, this platform has the ability to simultaneously concentrate and separate multiple analytes. This enables users to obtain simultaneous measurements of multiple cellular kinase activities that could reveal important information about their functional relationships.by Lih Feng Cheow.Ph.D

Development of an ELISA with Enhanced Sensitivity in a Nanofluidic System

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2009.Cataloged from PDF version of thesis.Includes bibliographical references (p. 66-68).Experimental studies were performed to evaluate the kinetics and equilibrium binding constants of biomolecules in nanofluidic channels. Binding events in the nanochannel were detected using electrical and fluorescence methods. We concluded that antibody-antigen binding constants in nanochannels were similar to experiments performed in microtiter plates at low antigen concentrations; however the bound fraction in nanochannels at high antigen concentration decreased due to steric hindrance. Binding kinetics in nanochannels was limited by convective transport of analytes, instead of diffusion or reaction. We also found that enzymatic reactions in nanochannels were very effective due to short diffusion length and high surface area to volume ratio. A bead based ELISA was developed to exploit the rapid binding reactions in the bulk and efficient enzymatic conversion in the nanochannels. Additionally, electrokinetic concentrators were integrated with multiplexed bead based ELISA to further improve the detection sensitivity of a sandwich immunoassay.by Lih Feng Cheow.S.M

Continuous Signal Enhancement for Sensitive Aptamer Affinity Probe Electrophoresis Assay Using Electrokinetic Concentration

We describe an electrokinetic concentration-enhanced aptamer affinity probe electrophoresis assay to achieve highly sensitive and quantitative detection of protein targets in a microfluidic device. The key weaknesses of aptamer as a binding agent (weak binding strength/fast target dissociation) were counteracted by continuous injection of fresh sample while band-broadening phenomena were minimized due to self-focusing effects. With 30 min of continuous signal enhancement, we can detect 4.4 pM human immunoglobulin E (IgE) and 9 pM human immunodeficiency virus 1 reverse transcriptase (HIV-1 RT), which are among the lowest limits of detection (LOD) reported. IgE was detected in serum sample with a LOD of 39 pM due to nonspecific interactions between aptamers and serum proteins. The method presented in this paper also has broad applicability to improve sensitivities of various other mobility shift assays.Singapore-MIT Alliance (Singapore MIT Alliance-II CE programme)Singapore. Agency for Science, Technology and Research (Scholarship

MEMS filter with voltage-tunable center frequency and bandwidth

This paper reports on the design of a reconfigurable ladder filter using RF MEMS resonators with voltage-tunable series and parallel resonance frequencies. The ladder filter consists of one shunt and two series resonators operating in the half-wave thickness shear vibration mode. It demonstrates a center frequency tuning range of 8 MHz at 817 MHz and an adjustable bandwidth from 600 kHz to 2.8 MHz, while maintaining an insertion loss \u3c 4 dB, stop-band rejection \u3e 30 dB and pass-band ripple \u3c 2 dB. This voltage tunable design enables channel agility and reconfigurability, substantially reducing the filter count in channel-select radio receiver architectures. Finally, a simple algorithm is provided to facilitate dynamic tuning of filter center frequency and bandwidth

High-throughput and massively-parallel concentrator for multiplexed immunoassays applications (다중 면역 결합 반응을 위한 고출력, 병렬 농축 장치)

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High throughput preconcentration device for immuno-binding application(면역 결합 반응을 위한 고출력 농축 장치)

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Dielectrically transduced single-ended to differential MEMS filter

Single-ended to differential micromechanical filters with large stop band rejection are ideal replacements for conventional SAW and FBAR filters [1,2] in sensor network transceivers and GSM and W-CDMA cell phones, which depend on differential signal paths. A differential output from the front-end filter eliminates the need for an off-chip balun in front-end radio design and increases filter linearity (Fig. 17.6.1). This paper reports on the design and performance of a single-ended input to differential output resonant electromechanical filter at 425MHz center frequency with 1MHz bandwidth (BW), 8dB insertion loss (IL), <5dB pass-band ripple,-50dB stop-band rejection, and-48dB common mode suppression (CMS), for a footprint of about 150×150µm 2. Fully differential mechanical filters can be operated in singleended to differential mode by providing only one of two input signal

Massively-parallel concentration device for multiplexed, high-throughput applications

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High-κ dielectrically transduced MEMS thickness shear mode resonators and tunable channel-select RF filters

Electrically coupled, high quality factor (Q), tunable channel-select ladder filters comprised of dielectrically transduced thickness shear mode resonators are presented using integrated circuit compatible bulk micromachining technology. The filters are fabricated on the 3.2 μm thick device layer of heavily doped SOI wafers with a 20 nm thick hafnium dioxide film sandwiched between the polysilicon electrodes and the silicon device layer. The ladder filter consists of shunt and series resonators operating in the half-wave thickness shear vibration mode. Dielectric transduction provides a k2 reduction in motional impedance relative to air-gap electrostatic transduction. Each constituent resonator of the filter can be excited at above 810 MHz resonant frequency with Q of 7800 in air and a motional impedance (RX) of 59 Ω. The ladder filter demonstrates a center frequency tuning range 8–817 MHz and an adjustable bandwidth from 600 kHz to 2.8 MHz, while maintaining an insertion lossdB, stop-band rejection \u3e30 dB and pass-band ripplefeature, RF MEMS filters can accommodate various signal waveforms with bandwidth range 0.1–5 MHz. In addition, errors due to fabrication can be compensated and capacitive loading in receiver architecture can be minimized