BioScript: programming safe chemistry on laboratories-on-a-chip

This paper introduces BioScript, a domain-specific language (DSL) for programmable biochemistry which executes on emerging microfluidic platforms. The goal of this research is to provide a simple, intuitive, and type-safe DSL that is accessible to life science practitioners. The novel feature of the language is its syntax, which aims to optimize human readability; the technical contributions of the paper include the BioScript type system and relevant portions of its compiler. The type system ensures that certain types of errors, specific to biochemistry, do not occur, including the interaction of chemicals that may be unsafe. The compiler includes novel optimizations that place biochemical operations to execute concurrently on a spatial 2D array platform on the granularity of a control flow graph, as opposed to individual basic blocks. Results are obtained using both a cycle-accurate microfluidic simulator and a software interface to a real-world platform

Optical Printing of Multiscale Hydrogel Structures

Hydrogel has been a promising candidate to recapitulate the chemical, physical and mechanical properties of natural extracellular matrix (ECM), and they have been widely used for tissue engineering, lab on a chip and biophotonics applications. A range of optical fabrication technologies such as photolithography, digital projection stereolithography and laser direct writing have been used to shape hydrogels into structurally complex functional devices and constructs. However, it is still greatly challenging for researchers to design and fabricate multiscale hydrogel structures using a single fabrication technology. To address this challenge, the goal of this work is the design and develop novel multimode optical 3D printing technology capable of printing hydrogels with multiscale features ranging from centimeter to micrometer sizes and in the process transforming simple hydrogels into functional devices for many biomedical applications. Chapter 2 presents a new multimode optical printing technology that synergistically combined large-scale additive manufacturing with small-scale additive/subtractive manufacturing. This multiscale fabrication capability was used to (i) align cells using laser induced densification in Chapter 3, (ii) develop diffractive optics based on changes in refractive indices in Chapter 4, (iii) print diffractive optical elements in Chapter 5, and (iv) digitally print complex microfluidic devices and other 3D constructs in Chapter 6. Overall, this work open doors to a new world of fabrication where multiscale functional hydrogel structures are possible for a range biomedical application

Continuous Nanoparticle Sizing and Characterization via Microfluidics

High-throughput manufacturing of nanomaterial-based products demands robust online characterization and quality control tools capable of continuously probing the in suspension state. But existing analytical techniques are challenging to deploy in production settings because they are primarily geared toward small-batch ex-situ operation in research laboratory environments. Here we introduce an approach that overcomes these limitations by exploiting surface complexation interactions that emerge when a micron-scale chemical discontinuity is established between suspended nanoparticles and a molecular tracer. The resulting fluorescence signature is easily detectable and embeds surprisingly rich information about composition, quantity, size, and morphology of nanoparticles in suspension independent of their agglomeration state. We show how this method can be straightforwardly applied to enable continuous sizing of commercial ZnO nanoparticles, and to instantaneously quantify the anatase and rutile composition of multi-component TiO2 nanoparticle mixtures pertinent to photo catalysis and solar energy conversion. A transport model of the interfacial complexation process is formulated to qualitatively confirm the experimental discovery and to provide understanding of the transport and binding processes. Practical utility is demonstrated by combining our detection method with a cyclone sampler to enable continuous monitoring of airborne nanoparticles. Our method uniquely combines ultra-high flow rate sampling (up to thousands of liters per minute) with sensitive detection based on localized fluorescent complexation, permitting rapid quantitative measurement of airborne nanoparticle concentration. By coupling these components, we show initial results demonstrating detection of airborne ultrafine Al2O3 nanoparticles at environmental concentrations below 200 μg m^−3 in air sampled at 200 L min^−1. This capability suggests potential for online monitoring, making it possible to establish dynamic exposure profiles not readily obtainable using current-generation personal sampling instruments. The underlying fluorescent complexation interactions are inherently size and composition dependent, offering potential to straightforwardly obtain continuous detailed characterization. The increasing commercial prevalence of nanoparticle-based materials also introduces a new demand for robust online characterization tools amenable toward online monitoring in manufacturing settings. We address this need by showing how electrical conductivity measurements can be exploited to instantaneously obtain size and species information in oxide nanoparticle suspensions. This approach is readily implemented in an easy to build platform that can be employed either online to provide real-time feedback during continuous synthesis and processing, or offline for evaluation of test samples obtained from larger batches. Our implementation enables accurate results to be obtained using inexpensive digital multimeters, suggesting broad potential for on-site deployment in industrial settings

Plasmonic Nanomaterials-Based Point-of-Care Biosensors

Point-of-care (POC) biosensors, although rapid and easy-to-use, are orders magnitude less sensitive than laboratory-based tests. Further they are plagued by poor stability of recognition element thus limiting its widespread applicability in resource-limited settings. Therefore, there is a critical need for realizing stable POC biosensors with sensitivity comparable to gold-standard laboratory-based tests. This challenge constitutes the fundamental basis of this dissertation work– to expand access to quality and accurate biodiagnostic tools. At the heart of these solutions lies plasmonic nanoparticles which exhibit unique optical properties which are attractive for label-free and labelled biosensors.Firstly, we improve the stability and applicability of label-free plasmonic biosensors for implementing biodiagnostics in POC and resource-limited settings. We demonstrate a cost-effective plasmonic paper-based biosensor for non-invasive detection of renal cancer. We also demonstrate a facile integration of plasmonic paper and microneedle patch to realize a POC biosensor which enables detection of target biomarkers present in interstitial fluid in an easy-to-use two-step process. We introduce a polymer encapsulation strategy to realize a stable and refreshable biosensor for long-term monitoring of protein biomarkers under harsh conditions. Next, we demonstrate dramatic improvement in bioanalytical parameters of POC biosensors by designing and realizing an ultrabright fluorescent nanolabel, plasmonic fluor. We discuss a novel approach for detection and quantification of inflammatory disease burden via plasmonically-active tissue analog which can undergo in vivo or ex vivo degradation in the presence of biological fluid associated with the tissue. We demonstrate a partition-free digital fluoroimmunoassay for ultrasensitive, multiplexed, and quantitative detection of protein biomarkers present in human biospecimens. Significantly, utilizing plasmonic-fluor, we overcome long-standing limitations associated with lateral flow immunoassays (LFA)– limited sensitivity, low accuracy and smaller analytical range compared to laboratory tests, and limited quantitation ability. Taken together, these advances are expected to overcome fundamental challenges associated with POC biosensors, and to bridge the gap between laboratory-based and at-home or point-of-care (POC) diagnosis. Through this dissertation work we demonstrate a complete workflow of a POC diagnostic platform that outperforms gold-standard laboratory tests in sensitivity, speed, dynamic range, ease of use, and cost

Establishment of a fully automatized microfluidic platform for the screening and characterization of novel Hepatitis B virus capsid assembly modulators

El procés de descobriment de fàrmacs s'enfronta a importants desafiaments a causa de la constant disminució dels guanys per medicament atesa la disminució en les noves aprovacions de la FDA combinada amb el constant augment dels costos i el temps de desenvolupament. Les plataformes integrades de detecció usant microfluídica van sorgir com a possibles solucions per accelerar el desenvolupament de molècules actives i reduir els requisits de temps i costos. El projecte VIRO-FLOW té com a objectiu identificar nous agents curatius per al virus de l'hepatitis B (VHB), integrant els avantatges de la química de flux continu amb tecnologies de bioassaigs in vitro en microfluídica. Durant aquesta tesi es va construir un sistema microfluídic aplicant dispositius modulars automatitzats. Es van redactar protocols d'avaluació per a les dades de fluorescència i reflexió, permetent el càlcul del factor Z, les desviacions estàndard, les corbes de dilució i els valors de concentracions efectives mitjanes màximes (EC50). La proteïna central del VHB (HBc) es va seleccionar com a objectiu principalEl proceso de descubrimiento de fármacos se enfrenta a importantes desafíos debido a la constante disminución de las ganancias por medicamento dada la disminución en las nuevas aprobaciones de la FDA combinada con el constante aumento de los costes y el tiempo de desarrollo. Las plataformas integradas de detección usando microfluídica surgieron como posibles soluciones para acelerar el desarrollo de moléculas activas y reducir los requisitos de tiempo y costes. El proyecto VIRO-FLOW tiene como objetivo la identificación de nuevos agentes curativos para el virus de la hepatitis B (VHB), integrando las ventajas de la química de flujo continuo con tecnologías de bioensayos in vitro en microfluídica. Durante la presente tesis se construyó un sistema microfluídico aplicando dispositivos modulares automatizados. Se redactaron protocolos de evaluación para los datos de fluorescencia y reflexión, permitiendo el cálculo del factor Z, desviaciones estándar, curvas de dilución y valores de concentraciones efectivas medias máximas (EC50). La proteína central del VHB (HBc) se seleccionó como objetivo principal.Drug Discovery as known today faces major challenges due to the constant decrease of earnings per drug given the decrease in new FDA approvements combined with the steadily rising development costs and time. Integrated microfluidic screening platforms emerged as possible solutions by accelerating the hit-to-lead development cycle and reducing time and cost requirements. The VIRO-FLOW project aims at the fast and efficient identification of novel curative agents for the Hepatitis B Virus (HBV), integrating the advantages of continuous flow chemistry with in vitro microfluidic bioassay technologies. During the present thesis a microfluidic system was built, applying automatized modular devices. Evaluation protocols were written for the fluorescence and reflection data, allowing the Z´-factor calculation, standard deviations, dilution curves, and half‐maximal effective concentrations (EC50) values. HBV core protein (HBc) was selected as primary target due to the ongoing demand for a functional cure to reduce the economic and social challenges imposed by the chronic diseas

Proceedings: Abstracts of Papers, 83rd Annual Meeting of the Virginia Academy of Science

Abstracts of the papers presented at the 83rd Annual Meeting of the Virginia Academy of Science, May 17-20, 2005, James Madison University, Harrisonburg, Virginia

Determination of epithelial growth factor receptor mutations in circulatory tumour cells from non-small cell lung cancer patients isolated using a novel microfluidic device

Patients with epidermal growth factor receptor (EGFR) sensitizing mutations in non small cell lung cancer (NSCLC) receive benefit from Tyrosine Kinase inhibitors. Accurate selection of patients before treatment is highly dependent on precise molecular diagnosis of EGFR mutations. Presently in the clinic, the diagnostic samples routinely used tumour biopsy and/or cell free DNA (cfDNA), are not sufficiently effective for precise diagnosis. Circulatory tumour cells (CTC) in blood have been explored successfully as alternative and complementary diagnostic markers to the current clinical tools. However, utility in the clinics has been hampered by the relatively low concentration of CTC in blood, and the lack of robust technologies that are adaptable for routine use. The present study describes the design and optimization of an immunomagnetic based microfluidic device (Lung card version II) that isolates CTC expressing the epithelial cell adhesion molecule (EpCAM) from blood with high capture efficiency and purity. The device is a 2-part system comprising a disposable chip that is simple in design and a reusable microfluidic unit that contains a mobile magnetic arm. The simple design and work-flow process of the device ensures cost efficiency for scalability and, ultimately, use in the clinic. The device was initially validated for its capability to isolate EpCAM positive cells. Results from spiking carboxylfluorescein succinimidyl ester stained EpCAM positive cells in media/blood showed a capture efficiency of ≥ 65% and a purity ≥ 97% from a 13ml sample in 50 minutes. The isolated CTC from NSCLC patients (n=38) were analysed for mRNA markers specific to malignant cells and were characterized for EGFR mutations following PCR and next generation sequencing. The mutational status of CTC was compared to that obtained from matched, tumour biopsy, samples. Significantly more mutations (P=0.0173) were detected in CTC enriched samples than the matched biopsy. Interestingly, mutations were detected in only 4 biopsy samples and the mutations detected in the biopsy were only concordant with results from CTC enriched samples for 1 patient. Exon 19 deletion was the most frequent mutation detected (86.7%) with rare mutations such as: L792P, C797S, H509R also been detected in CTC, and the present study reports the detection of K708R mutation in NSCLC for the first time. The clinical outcomes of patients who were positive for EGFR mutation from CTC, but had been placed on therapies based on mutation results from tissue biopsy were evaluated in this study. The results showed that no significant progression free survival (PFS) benefit was attained when comparing treatment response between patients whose CTC possessed an EGFR mutation and patients whose CTC possessed no EGFR mutation (10 months vs26 months p value-0.3420 HR- 0.76 95% CI- 0.2498-2.319). In summary the results from this study showed that the microfluidic device captured CTC with efficiency equal to other immuno-affinity based devices but had better purity rates and throughput and also that the device can be utilized for CTC processing for downstream analysis. Results from this current study further demonstrated the clinical potential of CTC+NGS matrix for the detection of EGFR mutations and the prospective impact it would have for precision oncology in NSCLC are discussed