Asghar, Waseem

Cerebrospinal fluid (CSF) has a role, in keeping the brain and spinal cord safe and nourished within the nervous system (CNS). This clear and colorless fluid is produced in the ventricles of the brain. Surrounds these structures acting as a protective cushion. CSF plays a role in maintaining nervous system health and ensuring optimal functioning. CSF accomplishes four objectives.
Protection: The brain and spinal cord are shielded from harm due to CSFs natural shock absorbing properties. This effectively safeguards these structures, from injuries caused by impacts or collisions.
Nutrition It ensures a favorable environment for neural cells to perform at their peak by supplying essential nutrients and removing waste products from the brain and spinal cord.

Heart failure is a chronic cardiovascular disease that is caused due to the lack of blood supply from heart. This lack of blood supply leads to accumulation of the fluid in the thoracic region. Traditionally, implantable cardioverter defibrillators (ICDs) are used to treat HF and to monitor its parameters. Healthcare wearable devices (HWDs) are healthcare devices that can be worn or attached to the skin. HWD are non-invasive and low-cost means of providing healthcare at the point-of-care (POC). Herein, this dissertation discusses the development of a HWD for the monitoring of the parameters of heart failure (HF). These parameters include thoracic impedance, electrocardiogram (ECG), heart rate, oxygen saturation in blood and activity status of the subject. These are similar parameters as monitored using ICD. The dissertation will discuss the development, design, and results of the HWD.

Plasma-based diagnostics are ideal for detecting a variety of diseases because they offer a method of detection that is minimally invasive, readily available, and easy to use for monitoring patients as they progress through a disease or respond to treatment. The only serum marker for PDAC is CA19-9 which lacks specificity, has limited sensitivity, and is unreliable for early detection. It is therefore of great importance to develop a diagnostic that is viable for screening and early detection. Exosomal miRNA were determined via bioinformatics analyses and then examined in PDAC cell lines to identify markers with greatest potential. These markers were then examined in plasma from PDAC patients and control groups. Four markers, miR-93-5p, miR-339-3p, miR-425-5p, and miR-425-3p, emerged as the most viable biomarker panel with the ability to detect PDAC in 100% of the early stages (N=5) compared to CA19-9 which showed increased levels in only one patient with early stage PDAC. Additionally, the diagnostic has a specificity of 80% and a sensitivity of 94.7%, making it comparable to CA19-9, and may even be beneficial for use in conjunction with CA19-9.
A plasma-based diagnostic was also developed for multi-strain HIV-1 detection utilizing the loop-mediated isothermal amplification (LAMP) method. LAMP primers were developed against the integrase and vpr regions of the HIV-1 genome. They were tested first in cultured HIV samples and then examined for their ability to amplify HIV-1 subtypes A-G. The integrase primer set provided a reliable means of diagnosing all 55 strains and isolates in under 30 minutes, whereas vpr was inconsistent and exhibited high variability in detecting the HIV subtypes. Our limit of detection for B-subtype with integrase was 30 viral copies/reaction. This could provide the basis for a novel, point-of-care diagnostic for use in underdeveloped regions.

In recent years, point-of-care (POC) microfluidic platforms have transformed the healthcare landscape as they offer rapid, low-cost, and easy operational benefits. POC diagnostics play an important role in expediting the testing process in resource-constrained areas. These platforms have become a powerful tool as they offer comparable results with gold-standard methods. The gold standard methods require sophisticated lab locations and expensive equipment, to process the samples which is a significant challenge particularly for people living in low-income countries. To address these limitations, herein, in my dissertation, I have developed POC microfluidic platforms that can be operated outside the laboratory using lesser equipment statistically hence reducing the testing cost and time. The developed POC chips are used for infectious diseases diagnosis for viruses such as Zika, Hepatitis C Virus (HCV), and severe acute respiratory syndrome coronavirus 2 (SARSCoV-2). The entire virus detection process was executed inside a uniquely designed, inexpensive, disposable self-driven microfluidic chip with high sensitivity and specificity. In addition to this, I have also developed a microfluidic platform for functional sperm cell sorting from raw semen samples. The microfluidic chip offers a platform where the sperm cells experience different shear stress in different parts of the chip that facilitates isolation of competent sperm cells without impacting their integrity. Simultaneously, it also allows effortless collection of sorted sperm cells from the collection chamber which holds clinical significance. All things considered, the developed devices are inexpensive, disposable, easy-to-use, and rapid that provide results within one hour.

The World Health Organization has identified the need for affordable, specific, rapid and deliverable point of care assays for infectious diseases in areas that are resource poor and lacking readily available complex testing methods.
The objective of this research is to discover improved methods of capturing and counting CD4+T, in a portable assay to aid in the detection of HIV or other diseases that are informed by cell identification and count.
The research divides into 4 major objectives: Design an improved portable, microchip. to isolate cells in a timely manner. Explore, design and prove the optical technology that provides large field-of-view and enables imaging large surface area simultaneously so that a sufficient sample can be collected. Test and analyze the microchip and optics to verify the specificity and efficiency of the biological process. Identify and count the cells in an automated manner.

Zika virus (ZIKV) is an emerging flavivirus transmitted to humans by Aedes mosquitos. ZIKV can be transmitted from mother to fetus during pregnancy and can cause microcephaly and other birth defects. Effective vaccines for Zika are yet to be approved. Detection of the ZIKV is based on serological testing that often shows cross-reactivity with the Dengue virus (DENV) and other flaviviruses. Currently, identification of ZIKV infection is usually done by i) testing the patient’s serum sample to detect ZIKV RNA using reverse transcriptase-polymerase chain reaction (RT-PCR), ii) testing patient’s serum sample for the presence of the NS1 protein antigen or iii) serological assays to determine the presence of virus-specific immunoglobin antibodies (IgG and IgM) by the use of ELISA assay. But ELISA-based assays show cross-reactivity and poor sensitivity. The gold standard for ZIKV RNA detection is RT-PCR, involves expensive medical facilities and skillful technicians. However, the plaque reduction neutralization test are executed to quantity neutralizing antibodies of the virus-but show high accuracy only after day 7 of the disease onset. Therefore, the development of POC assays which has the ASSURED (affordability, sensitivity, specificity, user-friendly, rapid and robust, equipment-free and deliverable) criteria defined by the World Health Organization are topmost priority. The core objective of this thesis is to find inexpensive, sensitive, precise, and fast assays for the specific diagnosis of ZIKV suitable for resource-constrained settings.

Genetic disorders like Cystic Fibrosis (CF) and X-linked Diseases (XLD) are inherited by offspring from parents who are healthy carriers of the autosomal recessive or allosomal genes. About 10-million Americans are healthy carriers of a mutant cysticfibrosis gene (predominantly F508del) and about 4% of newborns are at risk of being born with an X-linked disease. The current clinically approved mitigation plan for preventing genetic disorders in newborns from “at-risk couples” is to consider Preimplantation Genetic Testing for Monogenetic diseases (PGT-M). PGT-M involves an invasive microsurgical procedure that requires the removal of cells from 3-5day old embryos.
To minimize this invasiveness, we proposed a less invasive approach to prevent genetic disorders in newborns by genotypically sorting sperm cells which may be used for fertilization events (IUI/IVF/ICSI) with specially characterized antigens on the sperm surface membrane. For the disease models being adopted in our study – CF and XLD; we utilized certain monoclonal antibodies (mab) to target the H-Y male antigen and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein which are both selectively expressed on the sperm surface.

The majority of Sickle Cell Disease (SCD) prevalence is found in Sub-Saharan Africa, where 80% of the world’s population who suffer from this disease are born. Due to a lack of diagnosis and early treatments, 50-90% of these children will die before they reach the age of five. Current methods used for diagnosing SCD are based on hemoglobin analysis such as capillary electrophoresis, ion-exchange high-performance liquid chromatography, and isoelectric focusing. They require expensive laboratory equipment and are not feasible in these low-resource countries. It is, therefore, imperative to develop an alternative and cost-effective method for diagnosing and monitoring of SCD. This thesis aims to address the development and evaluation of a smartphone-based optical setup for the detection of SCD. This innovative technique can potentially be applied for low cost and accurate diagnosis of SCD and improve disease management in resource-limited settings where the disease exhibits a high prevalence. This Point-of-Care (POC) based device offers the potential to improve SCD diagnosis and patient care by providing a portable and cost effective device that requires minimal training to operate and analyze.

A cerebrospinal fluid (CSF) shunt system is used for treatment of hydrocephalus and abnormal intracranial pressure (ICP) conditions. Mostly a shunt system is placed under skin for creating a low resistance pathway between intracranial space and appropriate discharge sites within body by doing so excess CSF volume can exit the intracranial space. Displaced intracranial CSF volume normally results in lowered ICP. Thereby, a CSF shunt can manage ICP. In a healthy person, normal ICP is primarily maintained by CSF production and reabsorption rate as a natural tendency of body. If intracranial CSF volume starts increasing due to under reabsorption, this mostly results in raised ICP. Abnormal ICP can be treated by discharging excess CSF volume via use of a shunt system. Once a shunt system is placed subcutaneously, a patient is expected to live a normal life. However, shunt failure as well as flow regulatory problems are major issues with current passive shunt systems which leaves patients with serious consequences of under-/over CSF drainage condition. In this research, a shunt system is developed which is resistant to most shunt-related causes of under-/over CSF drainage. This has been made possible via use of an on-board medical monitoring (diagnostic) and active flow control mechanism. The developed shunt system, in this research, has full external ventricular drainage (EVD) capability. Further miniaturization will make it possible for an implantable shunt.

This thesis aims to address the challenges of the development of cost-effective and rapid assays for the accurate counting of CD4+ T cells and quantification of HIV-1 viral load for resource-constrained settings. The lack of such assays has severely affected people living in disease prevalent areas. CD4+ T cells count information plays a vital role in the effective management of HIV-1 disease. Here, we present a flow-free magnetic actuation platform that uses antibody-coated magnetic beads to efficiently capture CD4+ T cells from a 30 μL drop of whole blood. On-chip cell lysate electrical impedance spectroscopy has been utilized to quantify the isolated CD4 cells. The developed assay has a limit of detection of 25 cells per μL and provides accurate CD4 counts in the range of 25–800 cells per μL. The whole immunoassay along with the enumeration process is very rapid and provides CD4 quantification results within 5 min time frame. The assay does not require off-chip sample preparation steps and minimizes human involvement to a greater extent. The developed impedance-based immunoassay has the potential to significantly improve the CD4 enumeration process especially for POC settings.