Engineering At-Home Dilution and Filtration Methods to Enable Paper-Based Colorimetric Biosensing in Human Blood with Cell-Free Protein Synthesis

Abstract

Diagnostic blood tests can guide the administration of healthcare to save and improve lives. Most clinical biosensing blood tests require a trained technician and specialized equipment to process samples and interpret results, which greatly limits test accessibility. Colorimetric paper-based diagnostics have an equipment-free readout, but raw blood obscures a colorimetric response which has motivated diverse efforts to develop blood sample processing techniques. This work uses inexpensive readily-available materials to engineer user-friendly dilution and filtration methods for blood sample collection and processing to enable a proof-of-concept colorimetric biosensor that is responsive to glutamine in 50 µL blood drop samples in less than 30 min. Paper-based user-friendly blood sample collection and processing combined with CFPS biosensing technology represents important progress towards the development of at-home biosensors that could be broadly applicable to personalized healthcare.

Engineering the Signal Resolution of a Paper-Based Cell-Free Glutamine Biosensor with Genetic Engineering, Metabolic Engineering, and Process Optimization

“…E. coli cell extract for cell-free protein synthesis reactions was prepared according to previously reported protocols [38,39,63], except in this work we used BL21-Star™ DE3 (Invitrogen, Carlsbad, CA, USA) cells, which we engineered in this work with ∆lac and ∆glnA frameshift mutations (Figure S6). All growth media were 2 × YT media supplemented with 1 g/L glutamine unless otherwise noted.…”

“…The PANOxSP cell-free protein synthesis reactions were prepared as previously reported [38,39]. Glutamine was omitted from the PANOxSP preparation and was added separately only where explicitly noted.…”

“…We previously reported a glutamine sensor using "cell-free" or in vitro protein synthesis, a technology capable of low-cost, shelf-stable, portable, non-invasive, and rapid biosensors [38,39]. The prior work had critical assay background limitations.…”

“…In the case of glutamine sensing, this false-positive limitation was partially overcome by engineering the sensor with methionine sulfoximine (MSO) to inhibit glutamine synthetase [38]. However, the assay readable window and signal resolution were limited by a glutamine assay background that caused false positive results [39].…”

Advancement in Paper-Based Electrochemical Biosensing and Emerging Diagnostic Methods

“…Colorimetric methods are usually less expensive and simpler to operate than electrochemical techniques, and they can detect a wide variety of analytes. Colorimetric paper-based diagnostics have an equipment-free read-out, but raw blood obscures a colorimetric response which has motivated diverse efforts to develop blood sample processing techniques [2].…”