GRF 2024 9043519 PI: Chia-Hung CHEN

Microfluidic Single-Cell Surface Anchored Immunosorbent Assay for High-Throughput Screening of Secretory Phenotypes Applied to Synthetic Biology

Researcher(s)

• Chia-Hung CHEN (Principal Investigator / Project Coordinator) Department of Biomedical Engineering
• Chuch Loo POH (Co-Investigator)

Description

Synthetic biology harnesses the power of natural microbes by re-engineering metabolic pathways to manufacture high-value chemicals. Through a design-build-test cycle paradigm, secretory phenotypes can be selected in a mutant library to perform directed evolution for biofabrication.Immunosorbent assayis a promising tool to analyze a wide range of chemical compounds with a high-flexibility, including proteins and small molecules to indicate different secretory phenotypes; however, the throughput is limited. Recently,microfluidic droplet technologywas investigated for high-throughput single-cell secretory screening. However, due to the necessity of custom fluorescence assays within the droplets, the assay flexibility was limited. In fact, only a few chemicals (such as the enzyme horseradish peroxidase, HRP) with the available fluorescence chemical sensors were demonstrated to show the cases. To extend the capability of droplet assays, RNA-aptamer sensors, such as spinach RNA-aptamers-in-droplets (RAPIDs), have recently been developed; thus, certain metabolic secretions can be measured, but the challenge of measuring different chemicals with commercial values for industrial applications remains. Moreover, the limited E. coli encapsulation rate in the droplets (~1-2%) causes remarkable disadvantages in screening throughput. Thus, it is imperative to develop a new single-cell assay toflexiblymeasure high-value chemical compounds in ahigh-throughputmanner for advanced biofabrication. Here, we will develop a novel microfluidic single-cell surface anchored immunosorbent assay by integrating animmunosorbent assayanddroplet technologytogether as auniversal approachto analyze different valuable metabolic chemical compounds, including proteins and small molecules produced by E. coli, via three steps. 1. The captures will be grafted onto the cell surface. The E. coli will be encapsulated in the droplets to capture their own secretions. 2. After that, the E. coli with captured secretions will be extracted from the droplets for fluorescence labeling to indicate their secretory profiles. 3. The fluorescence-tagged E. coli will be screened via flow cytometry for secretory phenotyping and sorting (~103cells per second). This technology will therefore exhibit the following unique advantages:high-flexibilityto measure value chemicals, including both macromolecules and small molecules, by antibody/aptamer binding and labeling as well ashigh-throughputscreening and sorting regardless of the limited cell encapsulation rate in the droplets. This research will be conducted in three sections to achieve E. coli secretion analysis and biofabrication.First,a microfluidic single-cell surface anchored immunosorbent assaywill be developed. The capture molecules will be synthesized to contain the following parts: a dibenzocyclooctyne (DBCO) group and a capturer part (antibody or aptamer). E. coli with a functional group (azido) in lipopolysaccharide (LPS) on the outer membrane will be prepared. The capturer configurations will be grafted onto the E. coli surface through copper-free click interactions (click chemistry) to capture the target molecules for the immunosorbent assay.Second,the flexibility of the assay to measure different secretions(proteins and small molecules) from single E. coli will be demonstrated. For protein assays, thedefensinsecreted by E. coli will be captured by correspondingantibodiesand will be labeled via fluorescence barcoded detection antibodies. The fluorescence-tagged E. coli will then be screened in a high-throughput manner via flow cytometry. In the case of small molecule analysis, anaringeninassay will be performed. Instead of using antibodies, the corresponding aptamers will be used to capture naringenins for assaying. E. coli with gene variants of 4-coumaroyl-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI) for naringenin production will be investigated to guide the cell manufacturing workflow.Third,the mutant library will be screenedto sort secretory E. coli for the biofabrication offlavonoid-related products. The mutant library will be constructed by mutagenesis plasmid transduction. The aptamer sequences against specific flavonoids will be identified via systematic evolution of ligands by an exponential enrichment platform (SELEX) to develop capturers. The sorted E-coils will be analyzed via single-cell sequencing to investigate the mutant genes associated with the production of flavonoid compounds, including naringenin, apigenin, eriodictyol, kaempferol, etc. The successful realization of cell surface-anchored immunosorbent assays will offer a flexible and high-throughput tool to screen secretory phenotypes for extended biofabrication of high-value chemicals towards advanced synthetic biology. 

Detail(s)