Microfluidic cartridge with integrated array of amorphous silicon photosensors for chemiluminescence detection of viral DNA

Portable and simple analytical devices based on microfluidics with chemiluminescence detection are particularly attractive for point-of-care applications, offering high detectability and specificity in a simple and miniaturized analytical format. Particularly relevant for infectious disease diagnosis is the ability to sensitively and specifically detect target nucleic acid sequences in biological fluids. To reach the goal of real-life applications for such devices, however, several technological challenges related to full device integration are still to be solved, one key aspect regarding on-chip integration of the chemiluminescence signal detection device. Nowadays, the most promising approach is on-chip integration of thin-film photosensors. We recently proposed a portable cartridge with microwells aligned with an array of hydrogenated amorphous silicon (a-Si:H) photosensors, reaching attomole level limits of detection for different chemiluminescence model reactions. Herein, we explore its applicability and performance for multiplex and quantitative detection of viral DNA. In particular, the cartridge was modified to accommodate microfluidic channels and, upon immobilization of three oligonucleotide probes in different positions along each channel, each specific for a genotype of Parvovirus B19, viral nucleic acid sequences were captured and detected. With this system, taking advantage of oligoprobes specificity, chemiluminescence detectability, and photosensor sensitivity, accurate quantification of target analytes down to 70 pmol L-1 was obtained for each B19 DNA genotype, with high specificity and multiplexing ability. Results confirm the good detection capabilities and assay applicability of the proposed system, prompting the development of innovative portable analytical devices with enhanced sensitivity and multiplexed capabilities

Process automation for analytical measurements providing high precise sample preparation in life science applications

Laboratories providing life science applications will gain on improved analysis´ efficiency and reliability by automating sample pretreatment. However, commercially available automated systems are especially suitable for the standardized MTP-format allowing for biological assays, whereas automating analytical sample pretreatment is still an unsolved challenge. Therefore, the purpose of this presentation is the design, the realization, and evaluation of an automated system that supplies multistep analytical sample pretreatment and high flexibility for easy upgrading and performance adaption

Single-Molecule Detection of Unique Genome Signatures: Applications in Molecular Diagnostics and Homeland Security

Single-molecule detection (SMD) offers an attractive approach for identifying the presence of certain markers that can be used for in vitro molecular diagnostics in a near real-time format. The ability to eliminate sample processing steps afforded by the ultra-high sensitivity associated with SMD yields an increased sampling pipeline. When SMD and microfluidics are used in conjunction with nucleic acid-based assays such as the ligase detection reaction coupled with single-pair fluorescent resonance energy transfer (LDR-spFRET), complete molecular profiling and screening of certain cancers, pathogenic bacteria, and other biomarkers becomes possible at remarkable speeds and sensitivities with high specificity. The merging of these technologies and techniques into two different novel instrument formats has been investigated. (1) The use of a charge-coupled device (CCD) in time-delayed integration (TDI) mode as a means for increasing the throughput of any single molecule measurement by simultaneously tracking and detecting single-molecules in multiple microfluidic channels was demonstrated. The CCD/TDI approach allowed increasing the sample throughput by a factor of 8 compared to a single-assay SMD experiment. A sampling throughput of 276 molecules s-1 per channel and 2208 molecules s-1 for an eight channel microfluidic system was achieved. A cyclic olefin copolymer (COC) waveguide was designed and fabricated in a pre-cast poly(dimethylsiloxane) stencil to increase the SNR by controlling the excitation geometry. The waveguide showed an attenuation of 0.67 dB/cm and the launch angle was optimized to increase the depth of penetration of the evanescent wave. (2) A compact SMD (cSMD) instrument was designed and built for the reporting of molecular signatures associated with bacteria. The optical waveguides were poised within the fluidic chip at orientation of 90° with respect to each other for the interrogation of single-molecule events. Molecular beacons (MB) were designed to probe bacteria for the classification of Gram +. MBs were mixed with bacterial cells and pumped though the cSMD which allowed S. aureus to be classified with 2,000 cells in 1 min. Finally, the integration of the LDR-spFRET assay on the cSMD was explored with the future direction of designing a molecular screening approach for stroke diagnostics

Point of Care Molecular Diagnostics for Humanity

Diagnostics of disease at POC (point of care) has been declared one of the Grand Challenge by the Bill and Melina Gates Foundation (BMGF). Infectious diseases constitute a major cause of disease burden and cause more than half a billion Disability-Adjusted Life Years (DALYs) and millions of deaths each year. They have an especially large effect on children under 5 years of age. We have analyzed data from the GBD 2010 (Global Burden of Disease) project to emphasize the damage caused by infectious diseases, and highlight the opportunity of using diagnostic tools to rapidly identify and treat diseases. To motivate the work of this thesis, we quantify the expected impact of appropriate diagnostic technologies. We have also analyzed the requirements that a diagnostic tool should meet to generate the maximal global impact. We present various existing TPPs (Target Product Profiles) from different organizations and suggest some additions to these existing TPPs. We explain the particular molecular pathology technologies which have the potential to allow deployment of functional products in the developing world for point-of-care pathogen detection, especially in low-resource settings. We perform a detailed analysis on existing polymerase chain reaction (PCR) systems and describe the problems caused with thermal performance and optical interrogation. We list the requirements that disposable cartridges for such instruments should meet and suggest a metal base design with polymer top. After detailed FEA simulations, we demonstrate that the thermal response can be modeled using a one-dimensional (1D) lumped element system. We show improvements in thermal response due to using a metal base and the effect of fluid height. We also performed thermal-structural simulations to quantify the stresses on the adhesive bonds of metal/polymer cartridges. Next, we explain fabrication of these cartridges. We show methods to dispense adhesive using a robot and a custom made jig to spread the adhesive during curing. The cartridge was tested with different PCR reagents and we obtained reaction efficiencies approaching those of the commercial real time PCR machines. Our fabrication technique is useful to join dissimilar materials and is production friendly. By developing custom software, we observed the cartridge performance in a continuous manner. We could see the thermal response of cartridges by continuous fluorescence monitoring, and used reflective aluminum which increase light collection efficiency. We then present a simple and robust new way for thermal cycling. Robust thermal cycling has been a major challenge conducting PCR, especially in point of care situations. Here, we suggest a contact cooling approach, in which the cartridge rests on a thin metal plate with an integrated thin heater constructed from flexible printed circuit board (PCB) material. We use a solenoid to move a metal plate to cool down the sample cartridge during cycling. The metal plate then rests on a larger heat sink to disperse the shuttled heat. Our design is dust and water proof and was verified on a bench-top prototype. A novel optical design for fluorescence detection during qPCR is also described. We suggest a lateral illumination waveguide geometry with prism coupling that eliminates lenses and is integrated into an injection molded cartridge. The light is homogenized using a light guide, and we quantify the sources of scattered stray light from the chamber edge by performing ray tracing simulations to optimize the precise geometry. The design is tolerant to misalignments and enables easy coupling of LED light into the chamber. As the light collection efficiency is high, the size of the chamber can be very small. We tested real PCR reactions using this concept and observed a rapid integration time, enabling very fast reading. Sample preparation has been another challenge for all point-of-care (POC) lab-on-chip devices for many years. Here, we propose a new design which is robust, fast, flexible and simple, and uses a sliding seal to move the collected sample between various reservoir chambers. The sample moves on a slider sandwiched between seals that shuttles a DNA binding membrane between different reactions. Thus, size and volumes of reagents can be increased without increasing dead volumes. This design is easily automated, and positive displacement of fluids can work with many reagents without worrying about their characteristics such as foaming. The speed of the sample preparation protocols is high and complex protocols can be ported on this design concept, which we tested on real clinical samples and obtained impressive results. We designed and injection molded devices to test and verify this concept. Finally, we focus on instrumentation and software required to allow our technology to be used at the POC. We describe our embedded electronics and describe the powerful micro-controller and various high performance ICs that are used to construct a fully functional for sample to answer instrument. We developed various versions of software. The developer software allows us to control our system and bench top setup. Our end user product includes a tablet and cell phone software interface. Software was developed for a windows 8 tablet, windows 8 phone and an Android based devices. To conclude, we very briefly describe the POC systems that are under development: A portable qPCR system with a separate cartridge design, and a universal sample to answer system that performs qPCR, sample preparation and sample to answer protocols in one box depending on the cartridge. As per best of our knowledge the cost of this technology is much lower than any other option in its class. The sample to answer instrument is expected to cost less than $500. The test cost is expected to be less than$5. The performance is not compromised. We hope that this work can help bring a transformative change in the practice of pathology especially in the developing world.</p

Improvement of fluorescence-based microfluidic DNA analyzers

A tremendous effort continues in the development of micro-total-analysis-systems; in support of this, many chemical passivation methods have been developed to enhance the biocompatibility of such microfluidic systems. However, the suitability of these passivation techniques to many fluorescence-based assays still remains inconsistent. This part of this work is focused on the performance of a third generation intercalating DNA dye when used within microfluidic devices treated with a select variety of passivating coatings. The results of these tests indicate that passivation coatings which are intended to shed DNA based on electrostatic repulsion will in fact imbibe the fluorescent DNA intercalating dye by the same mechanism. Blocking this charge-based dye adsorption, such as with bovine serum albumen (BSA), has yielded mixed results in the literature. As an alternative, this present work indicates that preloading the bio- passivated microchannel with a small amount of this dye will prevent both the DNA and the DNA dye from being adsorbed from solution onto the channel walls. By characterizing the saturating behavior of this preloaded dye, a protocol is here suggested to optimize dye performance in passivated microfluidics. Furthermore, the intent and achievement of this work has been to design a BSA-free treatment method, thereby eliminating common fluorescent artifacts. The amount of dye preloading required is found to be proportional to the microchannel surface area, and can be predicted by a new material property defined for each chemical coating processes. Theoretical and experimental results indicate that this is independent of operating temperature, flow rate, and channel aspect ratio. Thus this is a property of the material, and not just a product of the several operational parameters. This property has been measured for four coatings as part of this work. Improvements in passivation are crucial to the development of lab-on-a-chip devices, important in solving current medical and healthcare problems. Challenging topics include the need for fast pathogen detection (i.e. ebola epidemic, HIV, water-borne diseases typhoid, cholera, dysentery) and the need for personalized medicine (i.e. cancer genomics, drug susceptibility). A novel microfluidic DNA analysis device was developed, incorporating helicase dependent amplification (HDA) and sequence-specific fluorescence based detection. These are incorporated into a glass/polymer platform that is hand operated and powered by a laptop computer. Thermal modeling sets operation at less than 3 Watts and fabrication consistency testing ensures samples volumes of 17µL. The heating element and optical components are powered via 3 USB ports. The heating element consists of a thin film heater and thermistor controlled in a feedback loop with Matlab and Arduino interfacing. Using fuzzy logic control, the temperature of the PCR chamber has been controlled by varying the heater voltage. On-chip amplification has been verified using a commercial LightScanner32 device and HDA detection and DNA melting analysis were performed on-chip

A wearable near-infrared diffuse optical system for monitoring in vivo breast tumor hemodynamics during chemotherapy infusions

Neoadjuvant chemotherapy (NAC) is increasingly being utilized to reduce tumor burden prior to surgery for breast cancer patients with stage II or higher disease. A pathologic complete response (pCR) to NAC has been correlated with longer 5-year survival and is generally considered as an absence of invasive cancer in the breast and axillary nodes at the time of surgery. Unfortunately, only about 10% of patients achieve pCR during NAC, and it may take months after the first infusion to determine response with methods that rely on anatomic information, such as palpation, mammography, ultrasound, and MRI. Functional imaging technologies such as Positron Emission Tomography, Magnetic Resonance Spectroscopy, and more recently, Diffuse Optical Spectroscopy, have shown promise for earlier predictions of therapy response. However, most of these techniques suffer from high expense, lack of portability, and safety issues related to the use of ionizing radiation or exogenous contrast agents. Furthermore, the repeated patient visits required by these techniques may hamper their clinical adoption for this purpose. This project aims to develop a new wearable diffuse optical device that can be used to investigate if very early timepoints during a patient’s first chemotherapy infusion are predictive of overall response (pCR versus non-pCR) to NAC. These timepoints correspond to an already scheduled patient visit and have so far been unexplored for their prognostic value. The development of this continuous-wave diffuse optical imaging device was conducted in three stages. First, a prototype rigid probe was designed and developed to test key optical and electrical components. Second, a high optode-density flexible probe was design and fabricated which can conform to the curved surface of the human breast. Finally, a control box with miniaturized electronics and high-speed electronics was designed and fabricated to complete a clinic-ready system. This system was then tested in both the laboratory setting and as part of a normal-volunteer clinical study in healthy subjects during a breath hold hemodynamic challenge.2019-10-22T00:00:00

Smartphone as a Portable Detector, Analytical Device, or Instrument Interface

The Encyclopedia Britannia defines a smartphone as a mobile telephone with a display screen, at the same time serves as a pocket watch, calendar, addresses book and calculator and uses its own operating system (OS). A smartphone is considered as a mobile telephone integrated to a handheld computer. As the market matured, solid-state computer memory and integrated circuits became less expensive over the following decade, smartphone became more computer-like, and more more-advanced services, and became ubiquitous with the introduction of mobile phone networks. The communication takes place for sending and receiving photographs, music, video clips, e-mails and more. The growing capabilities of handheld devices and transmission protocols have enabled a growing number of applications. The integration of camera, access Wi-Fi, payments, augmented reality or the global position system (GPS) are features that have been used for science because the users of smartphone have risen all over the world. This chapter deals with the importance of one of the most common communication channels, the smartphone and how it impregnates in the science. The technological characteristics of this device make it a useful tool in social sciences, medicine, chemistry, detections of contaminants, pesticides, drugs or others, like so detection of signals or image

Ensuring system integrity and security on limited environment systems

Cyber security threats have rapidly developed in recent years and should also be considered when building or implementing systems that traditionally have not been connected to networks. More and more these systems are getting networked and controlled remotely, which widens their attack surface and lays them open to cyber threats. This means the systems should be able to detect and block malware threats without letting the controls affect daily operations. File integrity monitoring and protection could be one way to protect systems from emerging threats. The use case for this study is a computer system, that controls medical device. This kind of system does not necessarily have an internet connection and is not connected to a LAN network by default. Ensuring integrity on the system is critical as if the system would be infected by a malware, it could affect to the test results. This thesis studies what are the feasible ways to ensure system integrity on limited environment systems. Firstly these methods and tools are listed through a literature review. All of the tools are studied how they protect the system integrity. The literature review aims to select methods for further testing through a deductive reasoning. After selecting methods for testing, their implementations are installed to the testing environment. The methods are first tested for performance and then their detection and blocking capability is tested against real life threats. Finally, this thesis proposes a method which could be implemented to the presented use case. The proposal at the end is based on the conducted tests

Doctor of Philosophy

dissertationThis project will produce an automated microfluidic system capable of extracting and purifying nucleic acids from raw samples for detection and analysis. The first step will be the development and characterization of microfluidic components and fabrication methods that will be implemented into the final device. The gas permeability properties of PDMS will be utilized to demonstrate integrated components for pumping, gas bubble trapping and removal, and enhanced mixing. Next, a three-layer PDMS with silicone membrane microfluidic platform will be developed to control fluid flow for nucleic acid purification processes. This microfluidic chip will be capable of taking a raw biological sample through the steps of cell lysis and solid phase nucleic acid extraction to deliver purified DNA or RNA for testing and analysis. The microfluidic chip will be mounted on a portable, desktop control system to allow automated device operation in clinics, laboratories, or the field. Finally, a disposable oscillatory flow PCR chip will be made from polycarbonate to amplify low concentrations of nucleic acid. The PCR module will also be controlled by the same instrument used for nucleic acid extraction. Temperature control will be provided by external heating blocks, and internal chip fluid temperature will be determined by numerical simulations. This device will be a step towards having a universal nucleic acid purification device to fill the much-needed niche in sample preparation for lab-on-a-chip applications