Porphyrin-modified antimicrobial peptide indicators for detection of bacteria

This study demonstrates the potential of porphyrin modified antimicrobial peptides for indication of bacterial targets on the basis of changes in the spectrophotometric characteristics of the construct. Detection is a result of changes in the structure of the antimicrobial peptide upon target binding. Those constructs comprised of peptides that offer little or no change in conformation upon interaction with bacterial cells demonstrated negligible changes in absorbance and fluorescence when challenged using Escherichia coli or Bacillus cereus. CD analysis confirms the presence/absence of conformational changes in the porphyrin-peptide constructs. Differing spectrophotometric responses were observed for constructs utilizing different peptides. The incorporation of metals into the porphyrin component of the constructs was shown to alter their spectrophotometric characteristics as well as the resulting absorbance and fluorescence changes noted upon interaction with a target. The described constructs offer the potential to enable a new type of biosensing approach in which the porphyrin-peptide indicators offer both target recognition and optical transduction, requiring no additional reagents

Synthetic beta cells for fusion-mediated dynamic insulin secretion

Generating artificial pancreatic beta cells by using synthetic materials to mimic glucose-responsive insulin secretion in a robust manner holds promise for improving clinical outcomes in people with diabetes. Here, we describe the construction of artificial beta cells (AβCs) with a multicompartmental 'vesicles-in-vesicle' superstructure equipped with a glucose-metabolism system and membrane-fusion machinery. Through a sequential cascade of glucose uptake, enzymatic oxidation and proton efflux, the AβCs can effectively distinguish between high and normal glucose levels. Under hyperglycemic conditions, high glucose uptake and oxidation generate a low pH (<5.6), which then induces steric deshielding of peptides tethered to the insulin-loaded inner small liposomal vesicles. The peptides on the small vesicles then form coiled coils with the complementary peptides anchored on the inner surfaces of large vesicles, thus bringing the membranes of the inner and outer vesicles together and triggering their fusion and insulin 'exocytosis'

Optofluidic Spectroscopy Platform for Detection of Hemolysis

In the United States alone, hundreds of millions of blood tests are performed annually, and a significant number of those tests are compromised due to hemolysis: e.g., 31% compromised in emergency rooms (inpatient) and 10% at blood banks, clinics, and other outpatient venues. Currently there is no way to reliably detect hemolysis without plasma separation. As a result, significant delays ensue, potentially negatively affecting patient diagnosis and treatment. In addition to in vitro hemolysis, which compromises the quality of blood tests, hemolysis can also occur in vivo. The in vivo occurrence of hemolysis is an indication of life-threatening complications. Being able to detect early signs of in vivo hemolysis would significantly improve outcomes for many patients, including pregnant women affected by HELLP (Hemolysis, Elevated Liver Enzymes, Low Platelet counts) syndrome. Therefore, there is a critical need to be able to detect hemolysis near the patient, immediately following the collecting of blood sample. The goal of this research is to provide an alternative to the traditional testing of blood samples, which requires large volumes of blood, centrifugation, and bulky instrumentation. The proposed alternative hemolysis detection system is a simple miniature setup that produces test results in minutes. This miniature, near-patient sensor would improve patients’ diagnosis, treatments, general satisfaction, and overall experience. The potential reduction of healthcare costs associated with hemolysis would be another significant benefit. The technology demonstrated in this dissertation is based on a novel combination of microfluidics, spectroscopy, and optical-fiber sensing. The microfluidics provide the capability to handle small volumes of liquid and to filter particles from solution. Novel membrane fabrication and modular integration provides the means to characterize and culture the captured particles. Spectroscopy and optical fibers provide the means to characterize the filtrate. These capabilities can be used for not only the detection of hemolysis but also other biomedical applications. . The first step in detecting hemolysis is to separate blood cells and other unwanted particulates from the plasma needed for optical analysis of concentration of hemoglobin. To that end, we focused initially on the problem of particle separation—specifically, within a microfabricated chamber with a custom-designed transparent membrane. To create a miniature microfluidic system capable of processing microliter blood samples, microelectromechanical systems (MEMS) fabrication techniques were required. The fabrication process included steps such as low-stress vapor deposition, photolithography, plasma, and wet etching. The resulting microdevice proved capable of filtering a variety of biological test fluids, including human lung fibroblast cancer cells from medium. The transparent membrane also allows for spectroscopic studies in broader applications, such as spectroscopic analysis or culturing of the cells retained on the filter. These capabilities were demonstrated using microbeads and cancer cells in solution. Optical techniques are used to analyze the separated blood plasma for concentration of hemoglobin. To integrate spectroscopic capabilities with the above microfluidics system, an optical fiber–based miniature probe was attached to the microfabricated chamber. As proof of concept, this system was tested in an application that required the measurement of physiologically relevant concentrations of cobalamin (vitamin B12). This application was used to address human error in drug administration showing measurements of cobalamin concentration as an example drug that can be monitored. The clinical means range of concentrations is from 1 µg/ml to 1000 µg/ml. The achieved results showed measurements of concentrations between 1 µg /mL to 5 mg/mL to monitor the physiological range and potential overdose in microliter of volume. This device has potential for numerous applications, ranging from single cell spectroscopy to measurements of glucose concentrations. This integrated system was then applied to the detection of hemolysis. The complete system conducts optofluidic spectroscopy with the optical fiber probe connected to the microfabricated chamber, which locally filters out blood cells, and reliably determine amount of free hemoglobin with the need for centrifuging. The utility of the device was demonstrated by its accurate measurement of hemoglobin concentrations in blood plasma. Finally, to apply the concept of the detection system to clinical condition with a reliable, and low-cost system, especially useful for developing countries, a smartphone-based technology, is proposed. This technology delivers ultra-fast results for the detection of early signs of HELLP syndrome and preeclampsia with the goal to decrease mortality and morbidity. The smartphone-based diagnostics is low cost, high speed of operation together with high accuracy. Detection of 1 mg/dL of free hemoglobin was achieved which is comparable to gold standard assay which are time consuming, difficult to operate and expensive. This technology, in summary, integrates microfluidics with microfiltration and spectroscopic technology to conveniently separate and characterize blood plasma. The device can also provide important information about other complex biological samples. These measurements require only very small sample volumes

Optofluidic Spectroscopy Platform for Detection of Hemolysis

In the United States alone, hundreds of millions of blood tests are performed annually, and a significant number of those tests are compromised due to hemolysis: e.g., 31% compromised in emergency rooms (inpatient) and 10% at blood banks, clinics, and other outpatient venues. Currently there is no way to reliably detect hemolysis without plasma separation. As a result, significant delays ensue, potentially negatively affecting patient diagnosis and treatment. In addition to in vitro hemolysis, which compromises the quality of blood tests, hemolysis can also occur in vivo. The in vivo occurrence of hemolysis is an indication of life-threatening complications. Being able to detect early signs of in vivo hemolysis would significantly improve outcomes for many patients, including pregnant women affected by HELLP (Hemolysis, Elevated Liver Enzymes, Low Platelet counts) syndrome. Therefore, there is a critical need to be able to detect hemolysis near the patient, immediately following the collecting of blood sample. The goal of this research is to provide an alternative to the traditional testing of blood samples, which requires large volumes of blood, centrifugation, and bulky instrumentation. The proposed alternative hemolysis detection system is a simple miniature setup that produces test results in minutes. This miniature, near-patient sensor would improve patients’ diagnosis, treatments, general satisfaction, and overall experience. The potential reduction of healthcare costs associated with hemolysis would be another significant benefit. The technology demonstrated in this dissertation is based on a novel combination of microfluidics, spectroscopy, and optical-fiber sensing. The microfluidics provide the capability to handle small volumes of liquid and to filter particles from solution. Novel membrane fabrication and modular integration provides the means to characterize and culture the captured particles. Spectroscopy and optical fibers provide the means to characterize the filtrate. These capabilities can be used for not only the detection of hemolysis but also other biomedical applications. . The first step in detecting hemolysis is to separate blood cells and other unwanted particulates from the plasma needed for optical analysis of concentration of hemoglobin. To that end, we focused initially on the problem of particle separation—specifically, within a microfabricated chamber with a custom-designed transparent membrane. To create a miniature microfluidic system capable of processing microliter blood samples, microelectromechanical systems (MEMS) fabrication techniques were required. The fabrication process included steps such as low-stress vapor deposition, photolithography, plasma, and wet etching. The resulting microdevice proved capable of filtering a variety of biological test fluids, including human lung fibroblast cancer cells from medium. The transparent membrane also allows for spectroscopic studies in broader applications, such as spectroscopic analysis or culturing of the cells retained on the filter. These capabilities were demonstrated using microbeads and cancer cells in solution. Optical techniques are used to analyze the separated blood plasma for concentration of hemoglobin. To integrate spectroscopic capabilities with the above microfluidics system, an optical fiber–based miniature probe was attached to the microfabricated chamber. As proof of concept, this system was tested in an application that required the measurement of physiologically relevant concentrations of cobalamin (vitamin B12). This application was used to address human error in drug administration showing measurements of cobalamin concentration as an example drug that can be monitored. The clinical means range of concentrations is from 1 µg/ml to 1000 µg/ml. The achieved results showed measurements of concentrations between 1 µg /mL to 5 mg/mL to monitor the physiological range and potential overdose in microliter of volume. This device has potential for numerous applications, ranging from single cell spectroscopy to measurements of glucose concentrations. This integrated system was then applied to the detection of hemolysis. The complete system conducts optofluidic spectroscopy with the optical fiber probe connected to the microfabricated chamber, which locally filters out blood cells, and reliably determine amount of free hemoglobin with the need for centrifuging. The utility of the device was demonstrated by its accurate measurement of hemoglobin concentrations in blood plasma. Finally, to apply the concept of the detection system to clinical condition with a reliable, and low-cost system, especially useful for developing countries, a smartphone-based technology, is proposed. This technology delivers ultra-fast results for the detection of early signs of HELLP syndrome and preeclampsia with the goal to decrease mortality and morbidity. The smartphone-based diagnostics is low cost, high speed of operation together with high accuracy. Detection of 1 mg/dL of free hemoglobin was achieved which is comparable to gold standard assay which are time consuming, difficult to operate and expensive. This technology, in summary, integrates microfluidics with microfiltration and spectroscopic technology to conveniently separate and characterize blood plasma. The device can also provide important information about other complex biological samples. These measurements require only very small sample volumes

Optofluidic spectroscopy integrated on optical fiber platform

Administering a wrong drug or a wrong dose can be extremely dangerous and can result in severe adverse effects or even the death of a patient. With human errors being possible, automatic real time identification of a drug and its concentration using technology is a viable option to decrease the chance of incorrect drug administration. As a step toward this goal, we propose a new optical fiber based spectroscopic system that has built-in filtration capabilities and thus can work in real time near patient without additional sample pre-processing. It is designed as a point probe consisting of an optical fiber with a miniature filtering reflector integrated on the interface. In the future it can be inserted into a bag for intravenous therapy (IV therapy) or in a syringe to measure the spectrum of the fluid and to confirm its properties. Additionally, use of microfluidic filtration allows to remove microscopic particles from the sample and thus decreases the noise and increases the sensitivity of spectroscopic measurement. In this study, an optofluidic system was fabricated, and filtration capabilities and measurement of cobalamin (vitamin B12) concentration have been demonstrated