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Electrochemical quantification of DNA using aluminum oxide membranes

Abstract DNA extraction automation is a major concern in molecular diagnostic where processing of numerous and daily samples of blood represent a labor-intensive task and are difficult to automate. With the rapid growth in the area of DNA diagnostics, there is an URGENT need for the development of a micro total analysis system which can do all the three main steps involved during nucleic acid-based diagnostics: sample preparation, extraction of the DNA, detection and quantification on one SINGLE chip. This work presents design, fabrication and testing of an integrated system, which can extract and electrochemically quantify DNA simultaneously from any unknown sample on one single chip. The system is fabricated using aluminum oxide membranes (AOM) as substrates for the extraction and quantification of DNA, bonded with the PDMS (Polydimethylsiloxane) block for providing the microfluidic inlet and outlet. Cost effective fabrication methods such as xurography (knife plotter) and soft lithography are used to obtain integrated hybrid fluidic and detector prototypes.

With the tremendous growth potential in this area, the trend is certainly in favor of miniaturization in the fields of genomics, proteomics and clinical diagnostics.
Since the mid 1990's the |iTAS technologies have exploded all across the world for biological sample analyses, chemical reactions, and diagnostics [3]. Low fluid volume consumption, faster analysis and response time, low fabrication costs leading to disposability, and compactness of the system are among a few of the benefits duly recognized to be significant.
Effective extraction and detection of biological molecules, for example, the extraction of DNA/RNA from blood cells, has become a core subject of interest for many researchers in the field of biology and life sciences. A variety of microfluidic DNA extraction approaches have already been developed based on different methods, such as those using silica beads [4], nanoporous membranes [5], magnetic silica particles [6], and chitosan-coated surfaces [7]. Out of these adopted procedures, nanoporous aluminum oxide membranes integrated with a microfluidic system has shown great results for extraction of gDNA from lysed whole blood [8].
Currently, detection and quantification of DNA is usually done by fluorescence detection, which has become a widely used technique in both laboratory scale and highthroughput genomic research [9]. However, electrochemical detection of DNA, when compared with optical detection, such as that of fluorescence detection, offers remarkable advantages such as: high sensitivity and selectivity, low cost, portability, label-free DNA detection, low-power requirements and miniaturization [10].
Despite substantial development in micro/nanofabrication technologies, there still exists the need for a microfluidic integrated system which can provide extraction, detection, and quantification of a genomic sample, like DNA, on one single chip.

Micro-Chip Based Detection Methods
The combination of microfluidics and lab on a chip, in the area of sensitive molecular detection, promises the production of small and low-cost analysis systems with improved sensitivity and selectivity, lower detection limits, and real-time analysis with ever-decreasing sample volumes. Notwithstanding the increased growth and development of microchip-based technologies over the last ten years, advances in effective and suitable detector systems for the detection and quantification of biological molecules has always lagged behind those of other process steps in the analysis of DNA [11].
To date, different modes of detection based on various detection concepts have been implemented for DNA analysis. All these concepts, and different methods based on these particular concepts, have been broadly classified in Figure 1.1.
The ultimate purpose behind this thesis is to design and fabricate a system that can extract unlabeled DNA using an aluminum oxide membrane (AOM) and simultaneously quantify the concentration of the DNA on the same chip. The main focus of this thesis will be on the detection and quantification of DNA using AOMs, as extraction of DNA using AOMs had been previously conducted successfully by our research group [12]. So far, many research groups have reported several detector systems for the detection of DNA using different detection concepts and principles. All these methods can be broadly distributed into two categories: labeling methods and label-free methods. The following section will serve as an overview of the various detector systems for detection and quantification of DNA that have been reported in the literature, featuring the optical, mass spectrometry and electrochemical readouts.

Optical Detection Systems
Optical detection of DNA and other biomolecules has found success employing laser-induced fluorescence (LIF) [13] and frequency modulated absorption. In LIF, DNA is detected with the use of fluorescent labeling [14], which has also been the most commonly used detection system for microchip-based applications. LIF detectors come with the advantage of high sensitivity and selectivity. Unfortunately, these are very expensive and the process is time consuming, though the use of the required bulky offchip control instrumentation is suitable for analytes that are naturally fluorescent, or molecules such as DNA that can be made highly fluorescent. The first fluorescence-based detector system was reported in 1995 by Krull [15]. This particular group used ethidium bromide as the indicator for DNA hybridization on optical fibers. Another group developed an on-chip fluorescence detector structure which uses a PINN+ photodiode with an interference filter and a robust liquid barrier layer [16]. With the application of photodiodes for DNA analysis, DNA was detected with a limit of 0.9 ng/^L using this system. Wolfgang Fritzsche, et al. [17], adopted the nanoparticle-labeling technique from microscope-based applications for DNA-chip detection.
With advantages like low background and high signal-to-noise ratios, and the other flexible properties of fluorescence such as wavelength and intensity, fluorescencebased detection methods are still dominating the field of detection for DNA analysis.
However, other factors such as the bulky control instrumentation, large sample requirements, and their high cost, tend to limit their miniaturization and portability.

Another interesting optics-based detection technique is Surface Plasmon
Resonance (SPR), in which label-free DNA can be detected. SPR-based sensors function by applying the principles of measuring the intensity [18], the resonant angle [19], and the wavelength of the reflected light [20]. One research group has shown work which focuses on dye-labeled DNA detection, using SPR-enhanced fluorescence readout [21].
Surface enhanced Raman spectroscopy (SERS) has also grabbed some attention for detection of biomolecules like DNA/RNA [22]. SERS is based on a surface-sensitive technique that can detect the enhancement in Raman scattering when molecules are adsorbed onto rough metal surfaces. Though providing high sensitivity and selectivity, the imaging system used in the sensors just discussed is prohibitively expensive and complex, limiting their application in p,TAS.

Mass-Sensitive Detection Techniques
Biomolecules have been detected and quantified in terms of their mechanical response to external forces. Different biomolecules like DNA/RNA, proteins, etc., are probed with the application of external force using such tools as optical tweezers [23] and magnetic beads [24]. Another approach in this area was reported by Lang, et al. in which they used direct transduction of DNA hybridization via surface stress changes into the displacement of microfabricated cantilevers, thus eliminating the need of labeling the DNA with fluorescent or radioactive tags. Adopting the same principle, another research group has achieved high-sensitivity DNA detection by fabricating an array of 33 micro cantilevers with SU-8 [25].
In these mass-based sensors, the transduction of DNA recognition or any other molecular recognition into a nanomechanical response is achieved by immobilizing a monolayer of receptor molecules on one side of the cantilevers and then detecting the mechanical bending induced by ligand binding in a liquid environment. Mass-sensitive detection sensors eliminate the need of using labeled DNA and bulky control instrumentation, and they are small and portable; however, the fabrication process for making these kinds of sensors is very convoluted and relies on classical photolithography techniques.

Electrochemical Detection Systems
For the last two decades, a wide range of electrochemical sensors for the detection of DNA have come to life and they continue to be an exciting field of research. The basic principle of an electrochemical detector system is to couple the biological recognition element (e.g., that of a DNA base-pair) to an electrode transducer, which then converts this particular recognition event to a useful electrical signal. These electrical signals, which can be in the form of current, voltage or impedance, can be measured to a fairly low limit, thus making a low detection limit for biomolecules feasible. Electrochemical sensors offer remarkable sensitivity and selectivity, without the rigorous sampleprocessing requirement of optical detector systems, label-free detection, low cost, simplicity, and portability, along with the inherent miniaturization of both the detector and control instrumentation. Thus, these sensors can be produced in mass quantities and be affordably disposable.
The history of electrochemical sensors for detection of DNA goes back to 1960, when Palecek first monitored the electrical activity of DNA by using oscillographic polarography with the application of mercury electrodes [26]. In another innovative approach, DNA oxidation was carried out through adsorption stripping voltammetry [27].
Since then, many other methods using silicon chips [28] and glass chips [29] as substrate for electrical detection of DNA have been discovered.
Electrochemical sensors can be divided into subcategories according to the electrochemical measurement technique used. These include measurement of current (amperometric sensors), current-voltage profile (voltammetric sensors), and voltage (potentiometric sensors). Amperometric and voltammetric sensors are most commonly used for detection and quantification of DNA. Their basic principle, advantages, and some of the state-of the art work done in this field are discussed next.

Amperometric Sensors
In amperometric sensors, a constant potential is applied across the working electrodes and the current associated with the reduction or oxidation of an electroactive molecule, involved in the recognition process, is monitored. In 1998, Wooley, et al.
carried out the first electrochemical detection of DNA on a glass chip, which was based on amperometric measurement, using integrated working and counter electrodes [30].
The working electrode was placed using photolithographic techniques just outside the exit of an electrophoresis channel, providing high sensitivity, while the reference electrode used was in the form of an Ag/AgCl wire. In related work, gold microelectrode surfaces were used for carrying out enzyme-based amperometric detection of DNA [31].
The numerous advantages of using electrochemical sensors based on amperometric detection include minimal dead volume, wide linear range, short response time, preparation of electrodes that can be miniaturized to both micro and nanoscale, and the method is also compatible with planar micromachining technology.

Voltammetric Sensors
In voltammetric sensors, current is measured as a function of applied voltage which is swept over a range [32]. For electrochemical detection, usually two kinds of voltammetry, namely linear scan voltammetry and cyclic voltammetry are used. In cyclic voltammetry an alternating triangular-wave voltage is applied at a fixed scan rate to the working electrode, in a three-electrode cell setup, whereas a linear potential ramp is applied for linear scan voltammetry. As discussed before, Palecek in 1988 performed DNA oxidation using adsorption stripping voltammetry. By using this technique, direct nucleic acid reduction was observed and the single-and double-stranded DNA were discriminated. More recently, a novel electrochemical sensor based on dynamic polymerase-extending hybridization for the detection of DNA was demonstrated [33] by the use of cyclic voltammetry, while in another study the catalytic square wave voltammetric detection of DNA was achieved with use of pyrolytic graphite electrodes [34].

Potentiometric Sensors
In potentiometric sensors, the molecular recognition process is converted into a potential signal, by which means the species of the analyte is obtained in connection with the use of ion-selective electrodes (ISE  [37]. In this work, they had developed CMOS compatible membranes using a high-energy electron beam and, using ionic conductivity measurements, they successfully demonstrated DNA detection. This was the first recorded use of an inorganic membrane for the purpose of discriminating DNA. In another study, modified nanoporous alumina has been used to detect the target DNA by monitoring the increase in the impedance at the electrode [38]. This group modified the membrane with covalently-linked single-strand DNA, which was then used for electrical detection of complementary target DNA sequences. Wanunu, et al. [39] have also shown detection of DNA-binding molecules using nanopores fabricated in ultrathin silicon membranes.

Motivation
With the rapid growth in the area of DNA diagnostics, there is an urgent need for developing a p,TAS which can carry out all three of the main tasks involved in DNA diagnostics, i.e. sample preparation or extraction of the DNA, detection, and quantification on a SINGLE chip. Combining these three processes would result in reduced specimen size, higher yield in extracted DNA, low cost, and affordable disposability with low environmental impact.
Nanoporous aluminum oxide membranes have already shown great potential in the biological and medical fields. Having already presented a successful demonstration of DNA extraction from lysed whole blood using these membranes, our research group, in this particular thesis, is presenting a microfluidics-based, label-free, and electrochemical DNA detection and quantification system based on aluminum oxide membranes. The use of electrochemical detection used in this system promises high sensitivity and selectivity, low cost, inherent miniaturization, portability and no use of classic photolithography techniques.

Outlines
The growth and development in the field of p,TAS and microfluidics has been presented in this chapter. It also discussed some of the state-of-the-art detection systems already developed by various research groups based on different detection concepts. The theory behind electrochemical detection and the importance of nanoporous aluminum oxide membranes will be discussed in Chapter 2. The basic equations used to describe the theory are taken from the previous literature. Design and fabrication of the DNA detection and quantification system will be explained in Chapter 3. The fabrication results and images of the fabricated system will be provided. This chapter will also contain the experimental methods for the quantification of gDNA based on an electrochemical detection concept. Chapter 4 contains the results of the optimization of the detection system. Key parameters, such as the effect of a change in the gap size between the electrodes, concentration of the DNA, effect of the voltage and the flow rate will also be investigated in that chapter. The last chapter will draw conclusions and summarize the experimental results in this thesis, and it will close with suggestions for future work in this area. help us better understand their interaction with DNA, so as to enable us to design our DNA quantification system.

Electrochemical Detection
This section describes the basic theory behind the electrochemical processes that occur during the detection of any particular sample. The electrochemical process is characterized by the transfer of electrons between the electrodes and molecules in the solution in proximity to the electrodes [40]. The fundamental reaction governing the science of electrochemistry is the equilibrium between two species, an oxidizing agent (Ox) that accepts electrons and a reducing agent (Red) that furnishes electrons, represented by the expression: During the detection of any nanoparticles or biomolecules such as DNA, it is usually assumed that the impedance of the sample to be detected is slightly different from the impedance of the buffer solution carrying this particular sample [41]. When a constant voltage is applied across the electrodes, it results in an electrochemical reaction causing electrochemically active molecules to be oxidized or reduced, which further results in a gradual change in the rate of transfer of electrons from one electrode to the other, thereby exhibiting a measurable variation in current flow through the circuit [42].
This change in the current can be measured either by measuring the current directly with an ammeter or a voltage drop across a series resistor.
Under ideal equilibrium conditions, electrochemical reactions are governed by the Nernst equation, which is used to determine the equilibrium reduction potential as RT fC0\ where, E0 is the standard potential for the redox couple, T is the Kelvin temperature, n is the number of electrons transferred in the reaction, F is the Faraday constant During the detection of the sample, change in the current (or impedance) in the detection area is possible because of three main reasons. These include: 1. If the sample being detected is electrically conductive, then an increase in current will be observed.
2. If the impedance of the sample being detected is more than the impedance of the bulk solution, then its presence will be signaled by a decrease in the current observed. A disturbance in the double layer will result in an increase of current and once the disturbance is eliminated, the current will drop again and the steady state will be reestablished. This concept of a double layer and the process of diffusion for mass transport making electrochemical detection possible is shown in Figure 2.1.
As summarized in Figure 2.1, an electrochemical reaction is comprised of many different processes that take place during the electrochemical process. When voltage is applied, the oxidized species in the bulk (O*) diffuses towards the electrode resulting in the transfer of its positive charge to form a reduced species (R). This reduced species with concentration CR diffuses away from the electrode and back into the bulk solution.
During this movement, the distance travelled by the redox molecule by diffusion can determined from Fick's law: where, x is the thickness of the double layer, D is the diffusion coefficient of the redox molecule and t is the time taken for the average molecule to travel.

Electrode Materials
During the electrochemical detection of DNA, the choice of the electrode material carries great importance. Various research groups have successfully demonstrated electrochemical activity of DNA using different electrode surfaces. Palecek studied the electrochemical behavior of DNA near mercury and mercury-amalgam electrodes [43] and showed that mercury electrodes can provide remarkable sensitivity for small changes in the DNA structure. With advantages like wide potential ranges, low electrical resistance, high resistance to corrosion and relatively low cost, carbon materials such as glassy carbon, carbon paste, carbon nanotubes, and graphite electrodes have been widely used for the electrochemical detection of DNA [44]. Gold electrodes have also been used for nucleic acid research, as they offer large double layer capacitance [45] and can be easily patterned with different gap sizes. Various other metals and metal oxides such as indium tin oxide, platinum, and silver have also been used in this area.

Anodized Aluminum Nanoporous Membranes
As the field of nanotechnology continues to grow rapidly, researchers in this field are able to characterize chemical, mechanical, and electrical properties down to the molecular level [46]. Applications of nanotechnology are versatile and one very relevant example of this field is the technology based on synthetic nanoporous membranes. These nanostructured materials have been used widely in different areas such as substrates for biological sensors [47], nano-filtration [48] , Synthetic nanoporous membranes can be divided into three different groups: isotropic (organic and inorganic), track-etch, and nanoporous anodic alumina membranes.
The main objective of this thesis is to use anodized aluminum nanoporous membranes or aluminum oxide membranes (AOM) as substrates for extraction, detection and quantification of genomic DNA. The following section will describe the manufacturing process of AOM and their advantages and limitations.
Nanoporous anodic alumina membranes were first reported in the year 1974 when the first patent on electrochemical reduction of aluminum was released [57] and since then, there has been a huge growth in the production of these membranes. Today, these membranes are commercially available from different companies with high open porosity, different pore diameters, and thickness. The pore aspect ratio (pore diameter vs. pore length) can reach up to values of 1:1000 and more [58]. During the anodization process of aluminum, two types of aluminum oxides can be produced on the surface [59].
If the process of anodization is carried out at a pH greater than 5, insulating films will be formed. However, if anodization is done in strong acidic electrolytes, then a film with pores open to the surface is formed which is known as 'anodized aluminum membranes'.
For the fabrication of these membranes, an aluminum foil with purity greater than 99.99 % is desired, which is annealed at 400 C for at least one hour, followed by electropolishing, commonly in a perchloric acid and ethanol solution. After this, anodization of the polished aluminum foil is done in a dilute acidic electrolyte (phosphoric, oxalic, or sulfuric acid) at a temperature below ~10 C. Once the anodization process is complete, then, using a galvanic process, the nonanodized aluminum is removed, and finally the closed ends of pores are opened using dilute phosphoric acid The pore diameter is dependent on two very important parameters: applied voltage and time for the anodizing step. High anodizing voltage results in larger pore diameters and can be varied between 1nm to 450nm with a narrow size distribution, a longer anodizing step will give more ordered pores and a large, thick alumina membrane.
Asymmetric pores, i.e., pores with different diameters can also be manufactured using two methods. The first method is to vary the voltage during the anodization step and the other method is to change the electrolyte solution during the growth process.  Conventional methods for DNA extraction systems are time consuming and complex and the DNA yield is very low, demanding large and expensive automated systems for sample preparation [62], [63]. Recently, our research group has shown successful demonstration of DNA extraction using a patterned aluminum oxide membrane [12]. In this particular work, we used commercially available AOMs, available from Whatman, Inc., UK, in three different pore sizes, 20nm, 100nm and 200nm.
All three pore sizes and three salt concentrations were tried to find an optimal physical and chemical condition for DNA extraction. The gDNA samples were prepared

Interaction of DNA with AOM
As discussed above, the pore size of an AOM is one of the most important parameters to be considered during the extraction and quantification of DNA. The collection rate of DNA for a given pore size is dependent on the pore shape and the aggregation of DNA. When analyte such as DNA binds with the walls of AOM, it has been observed that there are electrostatic interactions between gDNA and the alumina structure, which has many hydroxyl groups on its surface (Figure 2    For the analysis of biomolecules such as DNA, RNA, etc., there is a growing need for shifting all the key processes from classical photolithography methods to some other alternatives. During the detection and extraction of DNA, there is a need for an effective system, which is small in size, portable, inexpensive (so as to be affordably disposable), less dead volume, lower in detection limits, and high yield with regard to the extracted DNA sample. In this thesis, we present an integrated DNA extraction and quantification system that offers all the above listed advantages without use of photolithography. The following part of this chapter focuses on the detailed design and fabrication of this system.

Substrate
Both borosilicate glass slides and completely etched printed circuit board (PCB), a double-sided copperclad fiberglass board, were used in this work. Two important things were considered during the selection of the substrate material, which are as follows.

Strength and Stability
During the fabrication of this device, a substrate was needed that could serve as a platform to which we could easily bond our AOM. There was no concern about the electrical or thermal properties of the substrate, though there was concern regarding the mechanical stability the substrate would provide under normal operation. For these reasons, PCBs and borosilicate glass slides were chosen with thickness of 1.77mm and 1mm, respectively. Before we bond the AOMs to these substrates, we need to provide an outlet for the waste flow leaving the AOMs. For this, a 3mm diameter was drilled with a laser-sighted drill press (GMC) through the PCB, and with a Dremel moto-tool and a diamond bit through the glass slide. Since the drilling of the hole in the PCB is more of a cinch and less time consuming, as opposed to cutting through glass, PCBs were chosen as the main substrate building-block material for most of our experiments. For fabrication of miniature devices, these PCBs were cut into small pieces with dimensions of 30*20mm using a band saw. Once the PCBs of desired dimensions with the drilled holes were ready as shown in Figure 3.1 (step 1), the copper-cladding was completely etched off of these boards on both sides by full immersion in a ferric chloride bath for 20 minutes.

Bonding Capabilities and Cost
The next main factors considered in the selection of the substrate material concerned the bonding capabilities with the nanoporous membranes and the cost of these chips. As discussed earlier, for an effective integrated DNA analysis system, it is very important to have a system that is cheap, portable and simple in terms of design and fabrication, and thus, can be used as disposable chips. Based on these factors, fiberglass boards and glass slides were selected for our substrate materials, which both made manufacturing affordable in the quantities needed as well as provided a solid platform for our experimental devices.

Patterning of Aluminum Nanoporous Membranes
Commercially available AOMs, distributed by Whatman, Inc., UK, were used as the filter material for extraction and quantification of gDNA. The precise non-deformable honeycomb pore structure and narrow pore size of these membranes provides high flow rates and less dead volume for our samples. These membranes are available in three nominal pore sizes of 20nm, 100nm, and 200nm, and three diameters: 13mm, 25mm, and 47mm. Earlier, in Chapter 2, we described the effect of different pore sizes on the yield of the extracted DNA and from those results it has been shown that the 100nm pore size maximizes the DNA extraction rate. As, in this work, an integrated DNA analysis system that can simultaneously do extraction and quantification is being fabricated, all the results were characterized with this 100nm pore size membrane. However, results were also obtained with the 200nm pore membranes, as described in Chapter 4, to see the effect of pore size on quantifying DNA during the electrochemical detection.
The primary challenge in working with these membranes was their handling during the patterning, metal deposition, bonding to the substrate and incorporation of these membranes with the microfluidic system. The thickness of these membranes is close to 60^m and they are so brittle and weak in nature, that they get easily damaged during fabrication of the system. To overcome this challenge, these membranes were handled with great care and a new method was developed for their patterning and integration within the microfluidic system. Contact of these membranes with monomers, adhesives, surfactants, or wetting agents was avoided during the fabrication process, to ensure they remained ultra-pure without necessitating their cleaning at any stage.
During the pattering of these membranes, the fabrication of the channel on this membrane was the first most important part of the whole system, as it not only acts as the insulating spacer between the two electrodes but also defines the extraction and quantification area. Using a knife plotter (FC5100A-75, Graphtec Inc.), a shadow mask with straight channels was cut on a Rubylith film, which consists of a UV opaque red emulsion on clear polyester backing without an adhesive. To see the effect of various geometrical dimensions on these systems, we cut the film three different ways, with widths of 50^m, 100^m, and 200^m, and attached these to different membranes electrostatically.

Electrode Materials
Once each membrane with the shadow mask was ready, the electrodes were fabricated on the membrane itself. These electrodes are required to be highly planar and conductive. Sputtering (TMV sputtering systems) was used to deposit gold metal on the membranes. Since, these membranes are so brittle and thin, they cannot be loaded directly in the sputtering chamber, as it will damage the membranes. For this reason, the patterned AOMs were taped to a glass slide that served as a platform during the metal deposition process. Gold metal was deposited at 90W with a process pressure of 10mT for 10 minutes, which produced a layer with a thickness of 250nm. After the metal deposition was completed, the membranes were carefully removed from the glass slides and the shadow masks were equally carefully peeled off from the membranes with tweezers. These AOMs with a narrow channel between the electrodes were inspected under an SEM (scanning electron microscope) to measure the channel width and to see the pores between the electrodes (Figure 3.2).

Membrane Bonding to the Substrate
Another challenge in the fabrication of the system was bonding of thin patterned membranes to the PCB or glass substrate. For this process an adhesive (Loctite 4011) was applied in small dots using a syringe needle behind the patterned membrane and afterwards this membrane was aligned with the PCB substrate in such a way that the channel between the electrodes on the membrane aligned with the center of the drilled hole on the PCB. Care was taken not to apply substantial pressure during the bonding, as it could easily break the membrane.
Once the channel was perfectly aligned and bonded with the hole on the PCB as shown in the fabrication flowchart (Figure 3.1, step 2), the system was kept in a clean box to avoid contamination of the channel from dust particles.

Inlet and Outlet Ports
An important and crucial part of this system was integration of the patterned membranes with the microfluidics to fabricate inlet and outlet ports. This was achieved by attaching aluminum membranes with a PDMS tape that was further bonded to a thick To make a PDMS slab with a thickness of 3mm, 30mL of bubble-free PDMS was poured into another small petri dish and placed in the oven at the same temperature and for the same time.
Once the PDMS/tape composite and the thick PDMS slab were completely cured, they were removed from the petri dish and were cut into small blocks with dimensions of 5mm*5mm using a sharp steel blade. To connect the surface of the membrane with the tubing through these PDMS layers, a 2mm diameter hole was cut in the PDMS tape as well as the thick PDMS slab, separately, using a coring tool. Corona discharge (Enercon Dyne-A-mite 3D Treater) with an output voltage of 15kV was used for bonding of PDMS tape to the thick PDMS slab. Both surfaces were exposed for 40 seconds to the corona discharge and the cored holes on the PDMS tape and the slab were aligned properly under the microscope, after which the complete block was baked in a 65°C oven for 40 minutes Once cured, the backing tape was removed from the PDMS tape using a pair of fine tweezers and the adhesive side of the complete PDMS block was bonded over the patterned membrane under the microscope in such a way as to allow the inlet port in the PDMS block to align accurately with the channel on the membrane. To make the outlet port, an identical block of PDMS tape and with a thick PDMS slab having a cored hole of same diameter was fabricated and attached to the bottom side of the drilled hole on the PCB substrate. Care was taken to align the inlet and outlet ports with each other using alignment marks. Finally, the integrated system was placed in a 65°C oven for 30 minutes, so as to increase the bond strength (Figure 3.1, step 3).

Electrical Connections to the System
After the integration of inlet and outlet ports with the patterned membrane, the next key step includes producing electrical connections to the system. The electrical connections were created by bonding thin wires with both electrodes on the membrane, using a two component conductive silver epoxy (MG chemicals). Further, to cure the epoxy, the system was placed on a 150°C hot plate for 15 minutes (Figure 3.1, step 4).

Experimental Setup
The experimental setup for the integrated DNA detector system characterization consists of two syringe pumps, sample injection units and a pressure gauge in addition to the just-fabricated integrated microsystem device. The required DC supply to the detector system was provided by a source meter (Keithley 2400), which was also able to measure current and resistance. The source meter was connected to the PC through a GPIB card, and data was collected and then analyzed using LABVIEW software (version 8. In this chapter, a detailed description of the fabrication of the integrated DNA extraction and quantification system using AOMs bonded to PDMS tape was provided.
The experimental scheme to test the fabricated system using different concentrations of DNA was described. In the next chapter important parameters associated with DNA quantification systems will be studied and different concentrations of DNA will be quantified.
Drilled hole in the PCB substrate Step 1: PCB substrate with a drilled hole in the middle -► Sputtered metal ■►Patterned AOM Step 2: Bonding of patterned aluminum oxide membrane to the substrate Thick PDMS * slab ►PDMS tape Step 3: Bonding of PDMS tape and slab to the aluminum oxide membrane Electrical connections Step 4: Connection of electrical wires with the electrodes

□ c
Outlet port for the waste solution Step 5: Fluidic connections to the integrated system

Experiment II: Experiments with Buffer Solution and DNA Samples
In contrast to the initial results that were obtained by just pumping DNA samples directly through the system, our second set of experiments was carried out in two steps.
In the first step, only the buffer solution, which was the mixture of TE and Triton-X 100, was allowed to flow through the system for approximately 5-  The graph showing the repeatability of the results with this experiment is discussed in Appendix A. In order to validate the results further, we conduct our third and last experiment.

DNA Samples and Buffer Solution
In the previous section, we used an experiment in which the buffer solution was pumped through without any DNA for a particular time period and then DNA was injected into that buffer solution. From the graphs, we could deduce that the increase in current did not occur until after the DNA molecules were injected into the solution, presumably due to the higher conductivity of the DNA. In order to further validate these results, a third and last experiment was taken, which we break down into three detailed steps. First step, buffer solution is pumped through at a similar flow rate as previously for about 2-3 minutes followed by step 2, the injection of DNA samples into the buffer solution for approximately 10 minutes. In the third step, buffer solution is again passed through the membrane without letting any DNA molecules into the system for 2-3 minutes. Now, if there is an increase in the current as a result of any salt concentration or any other particles, then the current should decrease to its saturation limit same as for the results that had been obtained for the buffer solution before. If the current falls to the previous levels before DNA is injected, then it is proven that that there are no DNA

Calibration Curve
The main objective of designing and fabricating this system was to extract a certain amount of gDNA on the surface of aluminum oxide membrane and quantify the extracted mass of gDNA. Quantifying the mass or concentration of gDNA can help in determining the amount of sample required during the amplification, which is typically the next step in a genetic analysis system. Quantification of the gDNA will also eliminate many steps required during PCR, thus saving time and resources. For obtaining the calibration curve, we calculated A/s from the graph presented in      only buffer solution (0ng/^L) was pumped through the system, followed by injection of DNA samples for 10-12 minutes into the system. After 10 minutes, buffer solution (0ng/^L) was again pumped through the system.  This system thus provides us with several advantages such as the simultaneous extraction and quantification of gDNA, low detection limit of DNA (3.3ng/^L), less sample volume (200^l), high sensitivity and selectivity, low cost, small size, fast analysis time (less than 10 minutes), portability and disposable chips and more yield in extracted DNA compared to other quantification systems. Overall, when implemented in the area of DNA analysis this system can prove to be highly functional and valuable.

Future W ork
This thesis work has demonstrated successful fabrication of an integrated system that can extract and quantify gDNA simultaneously from any unknown sample. In the future, the experimentation would move to a higher realm of achievement while quantifying gDNA from blood samples, which will certainly save time and other resources. Other applications of this system include successful integration with a genetic analysis system. With different pore sizes of AOMs connected in series or parallel on one chip, this system can also be used for extracting and quantifying different biomolecules such as RNA, proteins and nanoparticles simultaneously.
Currently, the main problem with this system is the brittleness of the nanoporous oxide membranes. Under high pressures or stretching of electrical wires, these membranes tend to fracture easily and once the membrane is broken, the whole system fails. In this system, the electrical connections are made directly from the membrane to the source meter. As an alternative to these direct connections, one could make electrical connections in two steps. In the first step, a positive photoresist coated PCB board, patterned with bond pads on that PCB could be prepared according to certain specifications and design. Secondly, after patterning, the membranes are to be bonded on that PCB and using epoxy one can connect the electrodes on the membrane with the bond pads on the PCB that can be further soldered to long electrical wires connected to the source meter. Finally, I would also suggest that integrating this system with pneumatic micro valves would further increase the portability of the system, thus revolutionizing the whole methodology of DNA extraction and quantification.