A micro-electrophysiological analysis system (-EPAS) using various microfabrication techniques for single cell study was developed. Conventional microfabrication techniques combined with plastic and polymer microfabrication techniques have been used to realize the system. The system is capable of performing patch clamp recording and whole cell electrical impedance spectroscopy (EIS) on a single cell. Methodologies for single cell manipulation were developed. The ion channel activities of primary cultured bovine chromaffin cells were measured in both the patch clamping mode and the whole cell EIS mode. Membrane capacitance of the chromaffin cell was calculated from these measurements. Increases in the capacitances were observed when certain ion channels were blocked using toxins. The dielectric properties of human breast cancer cell lines from different pathological stages were measured and compared to a normal human breast cell line in the whole cell EIS mode. The measured properties were correlated to the pathological stages of the breast cancer cell lines. Decreases in the membrane capacitances were observed for the more pathologically progressed cancer cell lines.

Ovarian cancer is a high mortality disease where early stage detection may have significant survival benefits. Promising next-generation non-invasive, biomarker-based screening modalities involve longitudinal monitoring of serum biomarkers and multi-marker panel detection. Here, rapid, sensitive, precise and multiplexable diagnostic platforms can facilitate biomarker validation along with early detection and screening, and this work attempts to exploit the programmable bio-nano-chip (p-BNC) immunosensor to address these specific translational needs in ovarian cancer. First, the p-BNC was adapted for Cancer Antigen 125 (CA125) quantitation, the current FDA standard, with prominent implications in novel early detection and screening modalities. Antibody pairs binding to distinct epitopes on CA125 were identified and the p-BNC operating variables (incubation times, flow rates and reagent concentrations) were attuned to deliver optimal analytical performance (inter- and intra-assay precision of 1.2% and 1.9% and Limit-of-Detection (LOD) 1.0 U/mL), competitive with current gold standards, but with a short analysis time of 43 minutes. Further validation of the system with advanced stage patient sera (n=20) demonstrated good correlation with 'gold standard' ELISA (R² = 0.97). Next, the p-BNC was adapted for concomitant analysis of CA125 and Human Epididymis Protein 4 (HE4), a novel multiplexed biomarker panel for early detection and screening. The HE4 immunoassay was developed to perform optimally with the 'rate determining' CA125 assay. Cross-reactivity analysis demonstrated high specificity multiplexing. The dose-response curves for the multiplexed CA125 and HE4 immunoassays were congruous with their singleplex counterparts with respective LODs of 0.51 U/mL and 4.18 pM and a total analysis time of 44 minutes. A small pilot scale clinical study was conducted to discriminate between surgically confirmed patient sera (n=8) and corresponding age-matched healthy controls (n=8) utilizing the multiplexed p-BNC, interpreted with a risk of ovarian malignancy algorithm. Successful discrimination was achieved between the groups with Receiver Operating Characteristic (ROC) curve AUC (Area Under the Curve) values of 1.00, 0.984 and 1.00 respectively for CA125, HE4 and the composite marker combination. Taken together, the analytical and clinical performance, multiplexing capabilities and the short turn-around times on the p-BNC offer methodological advancements over current gold standard techniques, indicating strong promise for ovarian cancer diagnostics. / text

Electrophoresis separation corresponds to the motion and separation of dispersed particles under the influence of a constant electric field. In molecular biology, electrophoresis separation plays a major role in identifying, quantifying and studying different biological samples such as proteins, peptides, RNA acids, and DNA. In electrophoresis separation, different characteristics of particles, such as charge to mass ratio, size, and pI, can be used to separate and isolate those particles. For very complex samples, two or more characteristics can be combined to form a multi-dimensional electrophoresis separation system, significantly improving separation efficiency. Much effort has been devoted in recent years to performing electrophoresis separations in microfluidic format. Employing microfluidic technology for this purpose provides several benefits, such as improved transport control, reduced sample volumes, simplicity of operation, portability, greater accessibility, and reduced cost. The aim of this study is to develop microfluidic systems for high-performance separation of biochemical samples using electrophoresis methods. The first part of the thesis concerns the development of a fully integrated microfluidic chip for isoelectric focusing separation of proteins with whole-channel imaging detection. All the challenges posed in fabricating and integrating the chip were addressed. The chip was tested by performing protein and pI marker separations, and the separation results obtained from the chip were compared with those obtained from commercial cartridges. Side-by-side comparison of the results validated the developed chip and fabrication techniques. The research also focuses on improving the peak capacity and separation resolution of two counter-flow gradient electrofocusing methods: Temperature Gradient Focusing (TGF) and Micellar Affinity Gradient Focusing (MAGF). In these techniques, a temperature gradient across a microchannel or capillary is used to separate analytes. With an appropriate buffer, the temperature gradient creates a gradient in the electrophoretic velocity (TGF) or affinity (MAGF) of analytes and, if combined with a bulk counter-flow, ionic species concentrate at unique points where their total velocity is zero, and separate from each other. A bilinear temperature gradient is used along the separation channel to improve both peak capacity and separation resolution simultaneously. The temperature profile along the channel consists of a very sharp gradient used to pre-concentrate the sample, followed by a shallow gradient that increases separation resolution. A simple numerical model was applied to predict the improvement in resolution when using a bilinear gradient. A hybrid PDMS/glass chip integrated with planar micro-heaters for generating bilinear temperature gradients was fabricated using conventional sputtering and soft lithography techniques. A specialized design was developed for the heaters to achieve the desired bilinear profiles using both analytical and numerical modeling. To confirm the temperature profile along the channel, a two-color thermometry technique was also developed for measuring the temperature inside the chip. Separation performance was characterized by separating several different dyes, amino acids and peptides. Experiments showed a dramatic improvement in peak capacity and resolution of both techniques over the standard linear temperature gradients. Next, an analytical model was developed to investigate the effect of bilinear gradients in counter-flow gradient electrofocusing methods. The model provides a general equation for calculating the resolution for different gradients, diffusion coefficients and bulk flow scan rates. The results indicate that a bilinear gradient provides up to 100% improvement in separation resolution over the linear case. Additionally, for some scanning rates, an optimum bilinear gradient can be found that maximizes separation resolution. Numerical modeling was also developed to validate some of the results. The final part of the thesis describes the development of a two-dimensional separation system for protein separation, combining temperature gradient focusing (TGF) and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in a PDMS/glass microfluidic chip. An experimental study was performed on separating a mixture of proteins using two characteristics: charge to mass ratio, and size. Experimental results showed a dramatic improvement in peak capacity over each of the one-dimensional separation techniques.

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Ongoing work in the laboratory of Professor Chris Backhouse is aimed at developing a portable and inexpensive lab on a chip instrument. A system capable of molecular biology protocols including sample preparation (SP), polymerase chain reaction (PCR), and melting curve analysis (MCA) would meet the requirements for point of care genetic analysis. The SP, PCR, and MCA modules were designed and tested on a standalone basis and then integrated for analysis of raw clinical samples. An automated XY stage was developed for magnetic bead-based DNA purification. In addition, a LED/CCD-based optical detection module was employed for real time PCR and MCA. Data analysis algorithms and protocols were implemented to remove noise and interpret data. This work culminated in proof of principle on-chip SP-PCR-MCA to detect ß2m DNA from human buccal cells in a modular and inexpensive system. / Biomedical Engineering

abstract: Filtration for microfluidic sample-collection devices is desirable for sample selection, concentration, preprocessing, and downstream manipulation, but microfabricating the required sub-micrometer filtration structure is an elaborate process. This thesis presents a simple method to fabricate polydimethylsiloxane (PDMS) devices with an integrated membrane filter that will sample, lyse, and extract the DNA from microorganisms in aqueous environments. An off-the-shelf membrane filter disc was embedded in a PDMS layer and sequentially bound with other PDMS channel layers. No leakage was observed during filtration. This device was validated by concentrating a large amount of cyanobacterium Synechocystis in simulated sample water with consistent performance across devices. After accumulating sufficient biomass on the filter, a sequential electrochemical lysing process was performed by applying 5VDC across the filter. This device was further evaluated by delivering several samples of differing concentrations of cyanobacterium Synechocystis then quantifying the DNA using real-time PCR. Lastly, an environmental sample was run through the device and the amount of photosynthetic microorganisms present in the water was determined. The major breakthroughs in this design are low energy demand, cheap materials, simple design, straightforward fabrication, and robust performance, together enabling wide-utility of similar chip-based devices for field-deployable operations in environmental micro-biotechnology. / Dissertation/Thesis / Additional Paper / M.S. Civil and Environmental Engineering 2011

abstract: Rapid and reliable separation and analysis of proteins require powerful analytical methods. The analysis of proteins becomes especially challenging when only small sample volumes are available, concomitantly with low concentrations of proteins. Time critical situations pose additional challenges. Due to these challenges, conventional macro-scale separation techniques reach their limitations. While microfluidic devices require only pL-nL sample volumes, they offer several advantages such as speed, efficiency, and high throughput. This work elucidates the capability to manipulate proteins in a rapid and reliable manner with a novel migration technique, namely dielectrophoresis (DEP). Since protein analysis can often be achieved through a combination of orthogonal techniques, adding DEP as a gradient technique to the portfolio of protein manipulation methods can extend and improve combinatorial approaches. To this aim, microfluidic devices tailored with integrated insulating obstacles were fabricated to create inhomogeneous electric fields evoking insulator-based DEP (iDEP). A main focus of this work was the development of pre-concentration devices where topological micropost arrays are fabricated using standard photo- and soft lithographic techniques. With these devices, positive DEP-driven streaming of proteins was demonstrated for the first time using immunoglobulin G (IgG) and bovine serum albumin. Experimentally observed iDEP concentrations of both proteins were in excellent agreement with positive DEP concentration profiles obtained by numerical simulations. Moreover, the micropost iDEP devices were improved by introducing nano-constrictions with focused ion beam milling with which numerical simulations suggested enhancement of the DEP effect, leading to a 12-fold increase in concentration of IgG. Additionally, concentration of β-galactosidase was observed, which seems to occur due to an interplay of negative DEP, electroosmosis, electrokinesis, diffusion, and ion concentration polarization. A detailed study was performed to investigate factors influencing protein DEP under DC conditions, including electroosmosis, electrophoresis, and Joule heating. Specifically, temperature rise within the iDEP device due to Joule heating was measured experimentally with spatial and temporal resolution by employing the thermosensitive dye Rhodamine B. Unlike DNA and cells, protein DEP behavior is not well understood to date. Therefore, this detailed study of protein DEP provides novel information to eventually optimize this protein migration method for pre-concentration, separation, and fractionation. / Dissertation/Thesis / Ph.D. Chemistry 2014

De senaste decennierna har en miniatyriseringstrend inom ingenjörsvetenskaperna blivit allt större. Komplexa maskiner eller processer skalas ner till en allt mindre skala. Det kan vara motorer som inte är större än 500 μm eller kemiska analyser som vanligtvis görs på en större laboratorieutrustning som nu går att utföra på en yta på ca 2x4 cm. En sådan utrustning som kan utföra kemiska analyser kallas ofta för ”Lab-on-a-Chip” (LoC) och innehåller kemiska kretsar som hanterar mikroflöden av analysvätskor. En del av dagens forskning för att ta fram nya LoC handlar om att möta ett behov av portabel, billig och snabb analysutrustning i utvecklingsländer. Dock finns ett problem med att få ut produkter på marknaden. De flesta LoC som presenteras i forskningsrapporter idag är tillverkade av polydimetylsiloxan (PDMS). Det är en elastomer som lämpar sig väl för småskalig prototypframställning, men är svår att producera i stor skala, dessutom krävs ofta extern utrustning för att vätskeflöde skall uppstå. Det finns även LoC i papper, vilkas porösa struktur möjliggör för spontan vätsketransport, wicking, utan extern utrustning. Dessa är billiga och har nått större framgång. Exempelvis är vanliga graviditetstest som går att köpa på apoteket ofta LoC i papper. Textiliers fukt- och vätskehantering är relevant för komfort, och för många beredningsprocesser. Exempelvis är wicking ett välstuderat område som det finns djup kunskap om i den textila sektorn. Denna kunskap kan utnyttjas för att skapa ett textilt LoC. Att använda textila tekniker innebär möjligheter att styra vätskeflödet med hjälp av garn med och utan wickingförmåga. Denna studie undersöker hur en vävs naturliga X-Y-system av varp- och väftgarner kan utnyttjas för att skapa en kontrollerad vätskestyrning, en textil kemisk krets. Arbetet har utgått från frågan om hur en väv kan konstrueras för att leda en vätska från ett varpgarn till ett väftgarn utan läckage i oönskad del av väven. Två olika garner valdes: ett monofilament av polyeten för de områden där vätskeledning ej var önskvärd och ett multifilament av Coolmax® polyester med god wickingförmåga där vätskan vara avsedd att transporteras. Tre parametrar testades; bindningen i de delar av väven som var avsedd för vätsketransport (önskad väg); bindningen där vätskan skulle övergå från ett varpgarn till ett väftgarn (vägskälet); och antalet wickande trådar (trådigheten). Åtta olika kombinationer avseende dessa parametrar testade. Samtliga parametrar hade signifikant inverkan på läckaget. Den konstruktion med minst läckage in i oönskad väg var den med bindning över två trådar i önskad väg, flotteringar i vägskälet och var tvåtrådig. Den framtagna vävens möjlighet att användas i en biosensor undersöktes genom ett försök att konstruera en elektrokemisk glukosmätare. Som elektroder valdes en silverbelagd polyamid. Vid preparering av elektroderna skedde en oväntad reaktion mellan det silverbelagda garnet och en av de ingående kemikalierna, prussian blue. Därför kunde ingen detektion av glukos ske. Det noterades även att den textila kemiska kretsens wickingförmåga försämrades då den utsattes för våta prepareringsprocesserna av elektroderna. Från experimentet med att konstruera en textil glukosmätare drogs slutsatsen att preparering av elektroderna bör ske innan invävning i den textila kemiska kretsen.

Thesis advisor: Thomas C. Chiles / Rapid diagnosis of infectious disease at the site of the patient is critical for preventing the escalation of an outbreak into an epidemic. This is particularly true for cholera, a disease known to spread swiftly within resource-limited populations. A device suited to point-of- care (POC) diagnosis of cholera must not only demonstrate laboratory levels of sensitivity and specificity, but it must do so in a highly portable, low-cost manner, with a simplistic readout. Here, we report novel proof-of-concept lab-on-a-chip (LOC) electrochemical immunosensors for the detection of cholera toxin subunit B (CTX), based on two nanostructured architectures: the gold dendritic array, and the extended core coax (ECC). The dendritic array has an ~18x greater surface area than a planar gold counterpart, per electrochemical measurements, allowing for a higher level of diagnostic sensitivity. An electrochemical enzyme-linked immunosorbant assay (ELISA) for CTX performed via differential pulse voltammetry (DPV) on the dendritic sensor demonstrated a limit-of detection of 1 ng/mL, per a signal-to-noise ratio of 2.6, which was more sensitive than a simple planar gold electrode (100 ng/mL). This sensitivity also matches a currently available diagnostic standard, the optical ELISA, but on a miniaturized platform with simple electrical readout. The ECC was optimized and explored, undergoing several changes in design to facilitate sensitive LOC electrochemical detection. The ECC matched the off-chip sensitivity towards CTX demonstrated by a previous non-extended core coaxial iteration, which was comparable to a standard optical ELISA. In contrast to the previous coaxial architecture, the ECC is amenable to functionalization of the gold core, allowing for LOC detection. ECCs were functionalized using a thiolated protein G, and CTX was detected via an electrochemical ELISA. While this work is ongoing, the ECC shows promise as a platform for LOC electrochemical ELISA. The ability to potentially meet POC demands makes biofunctionalized gold dendrites and ECCs promising architectures for further development as LOC sensors for the detection of infectious disease biomarkers. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.