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This study developed cell-free biosensors targeting CA-125 for endometriosis and miRNA-29a for colorectal cancer to enable affordable, minimally invasive screening. The researchers assembled riboswitch and toehold switch reporters in E. coli and tested them in a cell-free expression system with GFP as the output signal. However, testing revealed inconsistent fluorescence where negative controls matched or exceeded positive reactions, indicating a failure to detect the target biomarkers reliably. Consequently, the authors plan to optimize reporter performance and confirm biomarker binding to develop functional point-of-care devices. This paper is centrally about endometriosis — specifically the development of a diagnostic biosensor using CA-125 as a target marker.
Abstract
Endometriosis and colorectal cancer are debilitating diseases lacking affordable, minimally invasive screening methods. Gold-standard diagnostics—laparoscopic surgery for endometriosis and colonoscopy for colorectal cancer—are invasive, expensive, and discourage early testing. We selected biomarkers CA-125, an elevated protein in endometriosis, and miRNA-29a, which is upregulated in colorectal cancer, to be the targets of our two biosensors: a riboswitch reporter to detect CA-125 and a toehold switch reporter based biosensor to detect miRNA-29a, with GFP expression as the output signal. We assembled plasmids containing our biosensors using Gibson assembly, transformed them into E. coli, and confirmed successful plasmid assembly via Sanger sequencing. Endometriosis biosensor testing in a cell-free expression system (CFES) showed inconsistent fluorescence where negative control reactions matched or exceeded the fluorescence of positive reactions regardless of biomarker concentration. In colorectal cancer biosensing, positive and negative reactions showed fluctuating, inconsistent fluorescence, showing no significant difference between the two conditions. To address this, we are also attempting to confirm miRNA is being expressed in the system or revise the biosensor design to increase GFP expression when miRNA-29 is present, as well as to confirm the binding of CA-125 to its attempted aptamer. In the future, we plan to optimize the performance of the reporters and develop them into portable & accessible point-of-care devices for practical use.
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Cell-Free Biosensors for Colorectal Cancer & Endometriosis Screening
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Abstract
Endometriosis and colorectal cancer are debilitating diseases lacking affordable, minimally invasive screening methods. Gold-standard diagnostics—laparoscopic surgery for endometriosis and colonoscopy for colorectal cancer—are invasive, expensive, and discourage early testing. We selected biomarkers CA-125, an elevated protein in endometriosis, and miRNA-29a, which is upregulated in colorectal cancer, to be the targets of our two biosensors: a riboswitch reporter to detect CA-125 and a toehold switch reporter based biosensor to detect miRNA-29a, with GFP expression as the output signal. We assembled plasmids containing our biosensors using Gibson assembly, transformed them into E. coli, and confirmed successful plasmid assembly via Sanger sequencing. Endometriosis biosensor testing in a cell-free expression system (CFES) showed inconsistent fluorescence where negative control reactions matched or exceeded the fluorescence of positive reactions regardless of biomarker concentration. In colorectal cancer biosensing, positive and negative reactions showed fluctuating, inconsistent fluorescence, showing no significant difference between the two conditions. To address this, we are also attempting to confirm miRNA is being expressed in the system or revise the biosensor design to increase GFP expression when miRNA-29 is present, as well as to confirm the binding of CA-125 to its attempted aptamer. In the future, we plan to optimize the performance of the reporters and develop them into portable & accessible point-of-care devices for practical use.
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