Periodic Reporting for period 1 - microPhage (Automated microfluidic phage display through non-fouling droplet-based technologies)
Periodo di rendicontazione: 2020-02-01 al 2022-01-31
Biosensors and in vitro diagnostics (IVDs) have revolutionized modern medicine by carving a path from primarily symptom-led diagnosis towards empirical and measurable biomarker-led diagnosis. The impact of these technologies is hard to overstate, and they have become an integral part of modern medical treatment pipelines, from initial diagnosis and prognosis through to treatment planning and monitoring. Biosensors and IVDs are ubiquitous within virtually every medical field, including infectious diseases, cardiology, oncology, and endocrinology. However, despite the ubiquity of these technologies, many challenges still remain. Biomedical research is continuously discovering novel and important biomarkers for disease, many of which could enable medical professionals to diagnose disease more accurately, and at an earlier stage. Unfortunately, newly discovered biomarkers are frequently present in the body in increasingly smaller concentrations, and existing technologies are inadequately equipped to detect them. Another major problem is the ever-present threat of disease mutations that can render existing technology useless; this is particularly problematic for infectious diseases. Finally, as diagnostic tests have become more routine, contemporary gold-standard technologies have begun to struggle with the demand. Given the importance of diagnostics, it is imperative that the global research community continues to address these issues through technological innovation. Key to this is the exploration and development of entirely new biosensing modalities that combine novel biochemical processes with powerful engineering solutions.
The primary objective of this project is to explore the interface of synthetic biology and microfluidics to generate new methods for detecting disease. I will develop a novel biosensing platform based on in vitro replication of cellular processes, and then combine this with the knowledge of the host lab to develop a state-of-the-art droplet microfluidic-based diagnostic platform. The platform will be assessed for analytical performance, and ultimately applied to the detection of several biomarkers for infectious diseases such as HIV. In addition to the primary objective, I anticipate that the results of this project will be of significant value to the global research community, and will inform the development of other projects at the interface of synthetic biology and microfluidics.
The primary objective of this project is to explore the interface of synthetic biology and microfluidics to generate new methods for detecting disease. I will develop a novel biosensing platform based on in vitro replication of cellular processes, and then combine this with the knowledge of the host lab to develop a state-of-the-art droplet microfluidic-based diagnostic platform. The platform will be assessed for analytical performance, and ultimately applied to the detection of several biomarkers for infectious diseases such as HIV. In addition to the primary objective, I anticipate that the results of this project will be of significant value to the global research community, and will inform the development of other projects at the interface of synthetic biology and microfluidics.
The work for this project was split into two areas; 1) synthetic biology / biochemistry and 2) microfluidic engineering. These areas were developed simultaneously and combined at the end.
Synthetic biology / biochemistry
My focus has been on developing the two enabling technologies for the biosensing platform; namely, a new technique for protein–DNA conjugation and the development of the cell-free biosensing machinery. Protein¬–DNA conjugation is necessary to couple the recognition of disease to the activation of the cell-free biosensing system. To this end, I achieved the following:
• Developed a novel method for attaching DNA to disease-targeting proteins using catalyst-free click chemistry. This method is able to furnish protein–DNA conjugates at a high conversion efficiency (95%) within 15 minutes.
• Developed several simple protocols for purifying protein–DNA constructs using common laboratory equipment, a problem which currently requires complex chromatographic setups.
• Developed electrophoretic and spectrometric protocols for analyzing and characterizing these constructs.
For the cell-free biosensing machinery, I achieved the following:
• Developed a protocol for generating the novel gene sequences required for programming the cell-free biosensor machinery and interfacing with the antibody¬–DNA conjugates. This protocol consists of multiple enzymatic DNA transformations and produces the desired genes in excellent purity (>99%).
• Showed that the protein–DNA conjugates are able to successfully activate in vitro cellular processes and initiate downstream fluorescent signaling pathways.
• Demonstrated that the signal generated from the interaction of the protein¬¬–DNA conjugates and the gene sequences is dependent on the concentration of the disease target. Thus, this approach represents a novel class of synthetic biology-drive disease biosensor.
Two manuscripts are being prepared to disseminate this work, one describing the preparation and characterization of the protein–DNA conjugates (“The Preparation and Analysis of Protein–DNA conjugates Furnished using Site-selective Click Chemistry”) and the other the preparation and function of the synthetic gene biosensors (“Combining Transcriptionally-repressed Synthetic Gene Circuits and Protein¬–DNA Conjugates to generate novel Synthetic-Biology Driven Biosensors”)
Microfluidic engineering
I have focused on exploring multiple avenues for partitioning the cell-free biosensor system into femtoliter volumes. This is necessary to create a highly automatable ultrasensitive digital diagnostic platform from the synthetic biology-driven biosensor I developed in parallel. I explored two routes for doing this – a droplet-based method and a glass microarray method. Broadly, I achieved the following objectives:
• Used soft lithography to produce a PDMS-based droplet generator that produces 20-micron diameter droplets
• Employed the developed droplet generator to encapsulate the cell-free biosensors I developed into droplets, and demonstrated successful biosensor function inside the droplets
• Designed and fabricated glass-based femtoliter arrays containing 50,000 individual chambers
This work is ongoing, and has formed the basis of several new PhD and Masters projects.
Synthetic biology / biochemistry
My focus has been on developing the two enabling technologies for the biosensing platform; namely, a new technique for protein–DNA conjugation and the development of the cell-free biosensing machinery. Protein¬–DNA conjugation is necessary to couple the recognition of disease to the activation of the cell-free biosensing system. To this end, I achieved the following:
• Developed a novel method for attaching DNA to disease-targeting proteins using catalyst-free click chemistry. This method is able to furnish protein–DNA conjugates at a high conversion efficiency (95%) within 15 minutes.
• Developed several simple protocols for purifying protein–DNA constructs using common laboratory equipment, a problem which currently requires complex chromatographic setups.
• Developed electrophoretic and spectrometric protocols for analyzing and characterizing these constructs.
For the cell-free biosensing machinery, I achieved the following:
• Developed a protocol for generating the novel gene sequences required for programming the cell-free biosensor machinery and interfacing with the antibody¬–DNA conjugates. This protocol consists of multiple enzymatic DNA transformations and produces the desired genes in excellent purity (>99%).
• Showed that the protein–DNA conjugates are able to successfully activate in vitro cellular processes and initiate downstream fluorescent signaling pathways.
• Demonstrated that the signal generated from the interaction of the protein¬¬–DNA conjugates and the gene sequences is dependent on the concentration of the disease target. Thus, this approach represents a novel class of synthetic biology-drive disease biosensor.
Two manuscripts are being prepared to disseminate this work, one describing the preparation and characterization of the protein–DNA conjugates (“The Preparation and Analysis of Protein–DNA conjugates Furnished using Site-selective Click Chemistry”) and the other the preparation and function of the synthetic gene biosensors (“Combining Transcriptionally-repressed Synthetic Gene Circuits and Protein¬–DNA Conjugates to generate novel Synthetic-Biology Driven Biosensors”)
Microfluidic engineering
I have focused on exploring multiple avenues for partitioning the cell-free biosensor system into femtoliter volumes. This is necessary to create a highly automatable ultrasensitive digital diagnostic platform from the synthetic biology-driven biosensor I developed in parallel. I explored two routes for doing this – a droplet-based method and a glass microarray method. Broadly, I achieved the following objectives:
• Used soft lithography to produce a PDMS-based droplet generator that produces 20-micron diameter droplets
• Employed the developed droplet generator to encapsulate the cell-free biosensors I developed into droplets, and demonstrated successful biosensor function inside the droplets
• Designed and fabricated glass-based femtoliter arrays containing 50,000 individual chambers
This work is ongoing, and has formed the basis of several new PhD and Masters projects.
The field of synthetic biology-driven biosensors is relatively nascent, only gaining traction within the last decade. To date, the vast majority of sensors have relied on translational control for detecting RNA targets. Whilst this has proven to be a powerful technique for detecting viral diseases, the fact that these sensors cannot be applied to protein targets is limiting. Protein-based biomarkers are still the dominant diagnostic marker for many diseases. By combining transcriptionally controlled synthetic gene circuits with protein–DNA conjugates, the technology I have developed enables these powerful tools to be applied to protein targets. This will greatly expand the market for this class of diagnostic biosensor and have potential implications across the entire field of in vitro diagnostics. Moreover, by developing novel methods for controlling cell-free machinery I anticipate that this work will inspire others to explore this emerging field and develop their own synthetic biology-driven biosensors. Finally, the work has provided fundamental value to the fields of protein modification and molecular biology. Protein–DNA conjugates are gaining traction as therapeutic tools, so novel ways to generate and analyze this class of molecules will be interesting to the pharmaceuticals industry. Similarly, studying the interaction between protein–DNA conjugates and synthetic cell-free gene networks could provide insight into the functions of cell-free machinery. This could in turn be used to inform the development of novel therapeutics. Given the influence that biological therapeutics (Biologics) have had on modern medicine, it is reasonable to expect that this work could have significant societal impact down the road.