Point-of-care biomarker quantification enabled by sample-specific calibration

Abstract

Easy-to-perform, relatively inexpensive blood diagnostics have transformed at-home healthcare for some patients, but they require analytical equipment and are not easily adapted to measuring other biomarkers. The requirement for reliable quantification in complex sample types (such as blood) has been a critical roadblock in developing and deploying inexpensive, minimal-equipment diagnostics. Here, we developed a platform for inexpensive, easy-to-use diagnostics that uses cell-free expression to generate colored readouts that are visible to the naked eye, yet quantitative and robust to the interference effects seen in complex samples. We achieved this via a parallelized calibration scheme that uses the patient sample to generate custom reference curves. We used this approach to quantify a clinically relevant micronutrient and to quantify nucleic acids, demonstrating a generalizable platform for low-cost quantitative diagnostics.

A primer on emerging field-deployable synthetic biology tools for global water quality monitoring

“…Of particular concern is the potential for other compounds present in environmental samples to interfere with biosensor components, or for organic matter to chelate contaminants and mask their presence. Thus, a major next step for biosensor development is to characterize these potential inhibitory effects and devise strategies to make biosensors robust against them 83 .…”

“…For example, a genetic system could be engineered to simultaneously detect multiple targets and produce an output that reports the identity and concentration of each target. Furthermore, new sample calibration strategies 83 are being developed to circumvent biosensors' intrinsic limitations and enable field-deployable sample quantification. As we continue harvesting parts from nature and clarifying biological design principles, we expect to see an improvement in the sensitivity and specificity of biosensors for an expanding list of detectable targets.…”

“…Biosensors also have the potential to detect emerging contaminants beyond arsenic and fluoride, including metals, agricultural products, and pharmaceutical and personal care products (PPCPs), such as antibiotics and cosmetics. Both whole-cell and cell-free biosensors have previously been used to detect metals by utilizing natural or engineered proteins; sensors have been reported for cadmium, lead, mercury, arsenic, copper, zinc, nickel, and cobalt, with sensitivities ranging from low parts per million to parts per billion 25,31,82,83 . Sensors for atrazine, a toxic herbicide, have also been developed by encoding a natural metabolic pathway for atrazine's conversion to cyanuric acid, which can be detected with a known protein sensor 84,85 .…”

Homogeneous and Universal Detection of Various Targets with a Dual‐Step Transduced Toehold Switch Sensor

“…It thus opens up a new sensing strategy with extraordinary potential for the development of easy, portable, or even point‐of‐care‐testing (POCT) assays. Previously, through the use of LacZ, green fluorescent protein (GFP), sortase, and alcohol acetyltransferase 1 (ATF1) as reporter proteins, real‐world Zika virus, gut microbiota, and norovirus RNA have been practically detected . Unfortunately, although such a promising function of the toehold switch sensor has been demonstrated, it generally requires in silico sequence design and laborious optimization of toehold switch sequence according to different individual targets .…”

Simple, Functional, Inexpensive Cell Extract forin vitroPrototyping of Proteins with Disulfide Bonds

“…Cell free protein synthesis (CFPS) has seen many recent applications in prototyping gene circuits, 1-3 sensors, [4][5][6][7][8] and therapies. [9][10][11] Cell extract derived from E. coli is the most widely used with efficient extract protocols, multiple supplement recipes, and clear experimental protocols available in literature.…”