Design and development of a lab-on-chip for biomedical analysis based on electrowetting on dielectric technique

The purpose of this thesis research project has been the development of a compact and versatile optoelectronic platform able to implement all the functionalities needed for a lab-on-chip operation. The project includes also the development of the electronics needed for the control of the system. In particular, the proposed platform includes three different modules designed for the fluid handling through the ElectroWetting On Dielectric (EWOD) technique, the thermal sample treatment and optical detection. These modules incorporate thin film microelectronic devices (such as photosensors and interferential filters for the optical detection, or heaters and temperature sensors for the sample treatments) on glass substrates connected to the electronic microcontrollers. Moreover, the use of handling techniques which avoid the use of pumps and syringes led to a portable, high-sensitive and low-power consumption lab-on-chip device. All of the modules have been designed, fabricated and tested separately. Finally, a device integrating all of the functionalities mentioned before has been designed for the development of a multifunctional platform able to perform a “true” lab-on-chip biomolecular system

Implementation of SELEX technique on Lab-on-Chip systems

The thesis presents the design and the experimental development of a compact and high sensitive Lab-on-Chip (LoC) system suitable for the implementation of SELEX technique. SELEX (Systematic Evolution of Ligands by EXponential enrichment) is a combinatorial technique used in molecular biology to produce copies of the same nucleotide and to select a strand of DNA (aptamer) specific for a target molecule. The proposed Lab-On-Chip system includes the following functional units: an amplification module based on the PCR technique; a separation module able to obtain a single strand DNA from a double strand DNA and a selection module for the specific selection of the aptamer. These functionalities are implemented combining microfluidic components (micro-channels and micro-valves), electronic devices (amorphous silicon photosensors, thin film heaters, temperature sensors) and bioanalytical procedure

Integrated sensor system for DNA amplification and separation based on thin film technology

This paper presents the development of a lab-on-chip, based on thin-film sensors, suitable for DNA treatments. In particular, the system performs on-chip DNA amplification and separation of double-strand DNA into single-strand DNA, combining a polydimethylsiloxane microfluidic network, thin-film electronic devices, and surface chemistry. Both the analytical procedures rely on the integration on the same glass substrate of thin-film metal heaters and amorphous silicon temperature sensors to achieve a uniform temperature distribution (within ±1 °C) in the heated area and a precise temperature control (within ±0.5 °C). The DNA separation also counts on the binding between biotinylated dsDNA and a layer of streptavidin immobilized into a microfluidic channel through polymer-brushes-based layer. This approach results in a fast and low reagents consumption system. The tested DNA treatments can be applied for carrying out the on-chip systematic evolution of ligands by exponential enrichment process, a chemistry technique for the selection of aptamers

An integrated microelectronic device for biomolecular amplification and detection

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2007.Includes bibliographical references (p. 133-154).The extraordinarily high sensitivity, large dynamic range and reproducibility of polymerase chain reaction (PCR) have made it one of the most widely used techniques for analyzing nucleic acids. As a result, considerable effort has been directed towards developing miniaturized systems for PCR, but most rely on off-chip optical detection modules that are difficult to miniaturize into a compact analytical system and fluorescent product markers that can require extensive effort to optimize. This thesis presents a robust and simple method for direct label-free PCR product quantification using a microelectronic sensor. The thesis covers the design, fabrication, and characterization of the sensing technique and its integration with PCR microfluidics into a monolithic detection platform. The sensor used in this thesis study is an electrolyte-insulator-silicon (EIS) device fabricated on planar silicon substrates. Based on electronic detection of layer-by-layer assembly of polyelectrolytes, the sensing technique can specifically quantify double-stranded DNA product in unprocessed samples and monitor the product concentration at various stages of PCR to generate readout analogous to that of a real-time fluorescent measurement.(cont.) Amplification is achieved with integrated metal resistive heaters, temperature sensors, and microfluidic valves. Direct electronic quantification of the product on-chip yields analog surface potential signals that can be converted to a digital true/false readout. A silicon field-effect sensor for direct detection of heparin by its intrinsic negative charge has also been developed. Detection of heparin and heparin-based drugs in buffer and serum has been studied, and a study demonstrating strong correlation between electronic heparin sensing measurements and those from a colorimetric assay for heparin-mediated anti-Xa activity has been performed.by Chih-Sheng Johnson Hou.Ph.D

Cryogenic deep reactive ion etching of silicon micro and nanostructures

This thesis focuses on cryogenic deep reactive ion etching (DRIE) and presents how it can be applied to the fabrication of silicon micro- and nanostructures that have applications in microfluidics and micromechanics. The cryogenic DRIE process relies on inductively coupled SF6/O2 plasma at temperatures below -100 °C. Low etching temperatures can cause some photoresist materials to crack, but Al2O3 has been shown to be a very well-suited masking material for cryogenic etching. The anisotropy of the etching process is enhanced by a thin passivation layer on sidewalls that prevents lateral etching. The main parameters that are used to adjust the thickness of the passivation layer are the process temperature and the O2 flow. Under adequate conditions vertical sidewalls are obtained, whereas passivation layers that are too thin result in negatively tapered sidewall slopes. Under conditions where a passivation layer is not formed, at higher temperatures and/or without oxygen flow, the etching profiles are isotropic. On the other hand, too high oxygen flow results in over passivation. Under conditions where the sidewall is slightly over passivated, its slopes are positively tapered, while more pronounced over passivation results in the formation of black silicon (or silicon nanograss, silicon nanoturf or columnar microstructures). Typically, vertical sidewall profiles are desirable. However, this thesis shall also demonstrate the usefulness of under and over passivation regimes. Here, highly anisotropic etching conditions are utilized to create trenches with vertical sidewalls, fluidic channels with regular micropillar arrays, and high aspect ratio silicon nanopillars. An isotropic etching process is utilized during the release of aluminum heaters fabricated on top of perforated free-standing Al2O3 membranes and silicon dioxide coated thermal silicon actuators. The fabrication process of three-dimensional sharp electrospray ionization (ESI) tips takes advantage of etching conditions that result in negatively tapered sidewalls. A self-feeding ESI interface for mass spectrometry (MS) is fabricated by combining a lidless micropillar filled channel with a sharp tip. Two approaches to the fabrication of silicon nanopillars are presented, both of which are suitable for wafer-scale manufacturing. One method combines silica nanoparticles with a highly anisotropic DRIE step, while the other method relies on highly over passivating conditions in a maskless DRIE process. Due to a large surface area and efficient light absorption in UV-range, silicon nanopillar structured surfaces are utilized as sample plates in laser desorption/ionization (LDI) MS. The wetting of nanopillar structured silicon surfaces is also studied. Fluoropolymer coated nanopillar structured surfaces have a contact angle of more than 170° and are ultrahydrophobic, whereas oxidized nanostructured surfaces are completely wetting. The accurate patterning of both completely wetting and ultrahydrophobic areas side by side allows complex droplet shapes and droplet splitters to be tailored

BioMEMS

As technological advancements widen the scope of applications for biomicroelectromechanical systems (BioMEMS or biomicrosystems), the field continues to have an impact on many aspects of life science operations and functionalities. Because BioMEMS research and development require the input of experts who use different technical languages and come from varying disciplines and backgrounds, scientists and students can avoid potential difficulties in communication and understanding only if they possess a skill set and understanding that enables them to work at the interface of engineering and biosciences. Keeping this duality in mind throughout, BioMEMS: Science and Engineering Perspectives supports and expedites the multidisciplinary learning involved in the development of biomicrosystems. Divided into nine chapters, it starts with a balanced introduction of biological, engineering, application, and commercialization aspects of the field. With a focus on molecules of biological interest, the book explores the building blocks of cells and viruses, as well as molecules that form the self-assembled monolayers (SAMs), linkers, and hydrogels used for making different surfaces biocompatible through functionalization. The book also discusses: Different materials and platforms used to develop biomicrosystems Various biological entities and pathogens (in ascending order of complexity) The multidisciplinary aspects of engineering bioactive surfaces Engineering perspectives, including methods of manufacturing bioactive surfaces and devices Microfluidics modeling and experimentation Device level implementation of BioMEMS concepts for different applications. Because BioMEMS is an application-driven field, the book also highlights the concepts of lab-on-a-chip (LOC) and micro total analysis system (μTAS), along with their pertinence to the emerging point-of-care (POC) and point-of-need (PON) applications

BioMEMS

As technological advancements widen the scope of applications for biomicroelectromechanical systems (BioMEMS or biomicrosystems), the field continues to have an impact on many aspects of life science operations and functionalities. Because BioMEMS research and development require the input of experts who use different technical languages and come from varying disciplines and backgrounds, scientists and students can avoid potential difficulties in communication and understanding only if they possess a skill set and understanding that enables them to work at the interface of engineering and biosciences. Keeping this duality in mind throughout, BioMEMS: Science and Engineering Perspectives supports and expedites the multidisciplinary learning involved in the development of biomicrosystems. Divided into nine chapters, it starts with a balanced introduction of biological, engineering, application, and commercialization aspects of the field. With a focus on molecules of biological interest, the book explores the building blocks of cells and viruses, as well as molecules that form the self-assembled monolayers (SAMs), linkers, and hydrogels used for making different surfaces biocompatible through functionalization. The book also discusses: Different materials and platforms used to develop biomicrosystems Various biological entities and pathogens (in ascending order of complexity) The multidisciplinary aspects of engineering bioactive surfaces Engineering perspectives, including methods of manufacturing bioactive surfaces and devices Microfluidics modeling and experimentation Device level implementation of BioMEMS concepts for different applications. Because BioMEMS is an application-driven field, the book also highlights the concepts of lab-on-a-chip (LOC) and micro total analysis system (μTAS), along with their pertinence to the emerging point-of-care (POC) and point-of-need (PON) applications

Polymer Pen Printing: A Tool for Studying 2D Enzymatic Lithography and Printing 3D Carbon Features

Polymer Pen Lithography (PPL) is a promising molecular printing approach which combines the advantages of both microcontact printing (low cost, high-throughput) and the dip pen lithography (DPN) (arbitrary writing, high-resolution) into one cohesive lithography method to create 2 dimensional (2-D) patterns with micro/nano-features on different substrates. The goal of this dissertation is to design and develop a new tool based upon PPL, which is not limited to forming 2D parallel patterns, but can also create 3D complex microstructures, finding applications in both biotechnology and Micro-Electro-Mechanical systems (MEMS) technology. This novel approach is named Polymer Pen Printing. Different from PPL using traditional dry-ink printing methods, an inking step is added to each printing repetition in the polymer pen printing process. Thus a wide range of ink materials with diverse viscosities can be transferred to substrates to create functional 2D and 3D microstructures. The polymer pen printing apparatus used in this thesis has been accomplished and introduced in Chapter 2. As a preliminary attempt, the single polymer pen printing approach was developed by simply attaching a solid polydimethylsiloxane (PDMS) pen tip to a multi-axis robot for small microarray fabrication. Compared to the single pen printing method, multi-pen printing can create large arrays of features. Therefore, an improved apparatus for polymer pen printing with high-throughput was discussed and built. Silicon molds, which consist of hundreds of uniform pyramidal openings, were photolithographically defined and etched using hydrofluoric acid (HF) followed by potassium hydroxide solution; after surface-modification with fluorosilane, these silicon molds were used to cast arrays of PDMS pyramidal pen tip. The cast PDMS pen array was mounted to a hollow holder with a 45° mirror inside. Therefore, each PDMS pen can be observed and monitored from the microscope on the side. To achieve prints less than 1 micron across, a Z axis stage with nanometer resolution was incorporated; and to control the compression of PDMS pen tips, a force gauge was also incorporated to detect 1 mg of applied force from the tips. The printing process for the multi-pen system is almost the same as single pen system. PDMS pens are coated with ink solution before each printing cycle by dipping into an inkwell and then brought into contact with the substrate surface. Thus multiple patterns, one from each tip, are created in parallel simultaneously. Furthermore, with control of the printing force, feature sizes could be controlled over the range submicron to tens of microns. Three ink candidates have been printed by polymer pen printing approach to fabricate 2D&3D microstructures. The first ink material is Barium Strontium Titanate (BST) nanocrystallites dispersed in a furfuryl alcohol (FA), which was printed by the single PDMS pen with 100 μm tip diameter (Chapter 3). After printing, samples were heated to crosslink FA monomers, forming a stable polymeric matrix with embedded BST nanocrystallites. Without shear-thinning properties, BST/FA ink cannot be used to build 3D posts, but it has the capability to create circular patterns with different thickness by the single or multi-tier deposition method. It was found that the thickness of film increased linearly with the number of deposits without changing the diameter significantly. This encouraging result could enable the formation of microcapacitors with multi-tiered structure. Moreover, the study of printing parameters, including printing height and ink pick-up position, shows that changes to the pen positions in the ink reservoir or substrate have essentially no impact on deposit thickness or diameter. Beyond that, the effect of surface chemistry of PDMS pen and silicon wafer have also been studied. The plasma treated hydrophilic PDMS pen can pen transfer more BST/FA than untreated one; and the larger diameters with smaller thickness were obtained on a hydrophilic silicon wafer. The second ink candidate is a dilute aqueous solution of enzyme Candia antartica lipase B (CALB), which is known to catalyze the decomposition of poly (ε-caprolactone) (PCL) films. By bringing enzymes into contact with pre-defined regions of a surface, a polymer film can be selectively degraded to form patterned features that are requited for applications in biotechnology and electronics. This so-called enzymatic lithography is an environmentally friendly process as it does not require any actinic radiation or synthetic chemicals to develop required features. But the need to restrict the mobility of the enzyme in order to maintain control of feature sizes poses a significant challenge. In Chapter 4, after writing 2D enzyme patterns onto a spin-cast PCL film by single pen printing, samples with CALB were incubated at 37 ℃ and 95% relative humidity (RH) for up to 7 days to develop features. The CALB selectively degraded the PCL film during incubation, forming openings through the film. The size of these features (10 to 50 μm diameter) is well suited for use as biocompatible micro-reactors. Previous study of patterning CALB by single polymer pen printing technique resulted in slow etch rates, low throughput and poor image quality. In Chapter 5, I present an improved enzymatic lithography approach, still based on enzyme CALB and PCL system, which can resolve fine-scale features (\u3c 1 μm across) in thick (0.1 - 2.0 μm) polymer films after 5 minutes to 2 hours of incubation at 37 ℃ and 87% RH. Immobilization of the enzyme on the polymer surface was monitored using fluorescence microscopy by labeling CALB with FITC. The crystallite size in the PCL films was systematically varied; small crystallites resulted in significantly faster etch rates (20 nm/min) and the ability to resolve smaller features (as fine as 1 μm). The effect of printing conditions and RH during incubation is also presented. Patterns formed in the PCL film were transferred to an underlying copper foil demonstrating a Green approach to the fabrication of printed circuit boards. In parallel, the third ink material is a mixture of 25 wt% graphite dispersed in a high viscosity phenolic resin n-methyl-2-pyrrolidone (NMP) solution, which can be converted into carbon/carbon composites after a pyrolysis process. The 3D polymeric posts were created by depositing multilayers of thixotropic phenolic ink on a silicon substrate by single polymer pen printing method with a 10 μm radius PDMS pen tip (Chapter 6). After pyrolysis at 1000 ℃ in a nitrogen (N2) atmosphere, the polymeric features were converted to the glassy carbon/graphite features with a high aspect ratio (\u3e2). These features may be used as microelectrodes. Last, arrays of needle-shaped glassy carbon have been developed by a drawing approach using multi-pen printing technique followed by simple pyrolysis process (Chapter 7). To build polymeric needles with ultra-high aspect ratio, the polymeric ink was prepared by dissolving phenolic resin in the high boiling point (204 ℃) solvent NMP without fillers to achieve good printability and suitable viscosity. By slowly lifting up the print head from substrate, liquid needle structures were formed and then solidified on silicon substrates or gold electrodes due to the solvent evaporation. In addition, suspended resin fibers connected to two electrodes have also been fabricated by precisely controlling the movement of the PDMS pen. After pyrolysis, these resin features were converted to glassy carbon and the 3D structures remained. The electrical characterization results showed that glassy carbon made by this method had relatively low resistivity (2.5 x 10-5 Ωm). Therefore the glassy carbon based microneedles are well-suited to be electrodes for electrochemical sensors for biological applications