Bacteria are an enormous and largely untapped reservoir of biosensing proteins. We describe an approach to identify and isolate bacterial allosteric transcription factors (aTFs) that recognize a target analyte and to develop these TFs into biosensor devices. Our approach utilizes a combination of genomic screens and functional assays to identify and isolate biosensing TFs, and a quantum-dot Förster Resonance Energy Transfer (FRET) strategy for transducing analyte recognition into real-time quantitative measurements. We use this approach to identify a progesterone-sensing bacterial aTF and to develop this TF into an optical sensor for progesterone. The sensor detects progesterone in artificial urine with sufficient sensitivity and specificity for clinical use, while being compatible with an inexpensive and portable electronic reader for point-of-care applications. Our results provide proof-of-concept for a paradigm of microbially-derived biosensors adaptable to inexpensive, real-time sensor devices.

A recent description of an antibody‐free assay is significantly extended for small molecule analytes using allosteric transcription factors (aTFs) and Förster resonance energy transfer (FRET). The FRET signal indicates the differential binding of an aTF–DNA pair with a dose‐dependent response to its effector molecule, i.e., the analyte. The new sensors described here, based on the well‐characterized aTF TetR, demonstrate several new features of the assay approach: 1) the generalizability of the sensors to additional aTF–DNA–analyte systems, 2) sensitivity modulation through the choice of donor fluorophore (quantum dots or fluorescent proteins, FPs), and 3) sensor tuning using aTF variants with differing aTF–DNA binding affinities. While all of these modular sensors self‐assemble, the design reported here based on a recombinant aTF–FP chimera with commercially available dye‐labeled DNA uses readily accessible sensor components to facilitate easy adoption of the sensing approach by the broader community.

Immobilization of biosensors in or on a functional material is critical for subsequent device development and translation to wearable technology. Here, we present the development and assessment of an immobilized quantum dot− transcription factor−nucleic acid complex for progesterone detection as a first step toward such device integration. The sensor, composed of a polyhistidine-tagged transcription factor linked to a quantum dot and a fluorophore-modified cognate DNA, is embedded within a hydrogel as an immobilization matrix. The hydrogel is optically transparent, soft, and flexible as well as traps the quantum dot−transcription factor DNA assembly but allows free passage of the analyte, progesterone. Upon progesterone exposure, DNA dissociates from the quantum dot−transcription factor DNA assembly resulting in an attenuated ratiometric fluorescence output via Forster resonance energy transfer. The sensor performs in a dose-dependent manner with a limit of detection of 55 nM. Repeated analyte measurements are similarly successful. Our approach combines a systematically characterized hydrogel as an immobilization matrix and a transcription factor−DNA assembly as a recognition/transduction element, offering a promising framework for future biosensor devices.

We describe an electrochemical strategy to transduce allosteric transcription factor (aTF) binding affinity to sense steroid hormones. Our approach utilizes square wave voltammetry (SWV) to monitor changes in current output as a progesterone (PRG) specific aTF (SRTF1) unbinds from the cognate DNA sequence in the presence of PRG. The sensor detects PRG in artificial urine samples with sufficient sensitivity suitable for clinical applications. Our results highlight the capability of using aTFs as the biorecognition elements to develop electrochemical point-of-care biosensors for detection of small molecule biomarkers and analytes. CONFLICT OF INTERESTKS, RB, CG, CMK, JEG, and MWG are inventors on a patent describing this technology filed by Boston University, which is available for license.

Immobilization of biosensors on surfaces is a key step toward development of devices for real‐world applications. Here the preparation, characterization, and evaluation of a surface‐bound transcription factor–nucleic acid complex for analyte detection as an alternative to conventional systems employing aptamers or antibodies are described. The sensor consists of a gold surface modified with thiolated Cy5 fluorophore‐labeled DNA and an allosteric transcription factor (TetR) linked to a quantum dot (QD). Upon addition of anhydrotetracycline (aTc)—the analyte—the TetR‐QDs release from the surface‐bound DNA, resulting in loss of the Förster resonance energy transfer signal. The sensor responds in a dose‐dependent manner over the relevant range of 0–200 µm aTc with a limit of detection of 80 nm. The fabrication of the sensor and the subsequent real‐time quantitative measurements establish a framework for the design of future surface‐bound, affinity‐based biosensors using allosteric transcription factors for molecular recognition.

Recently,a llosteric transcription factors (TFs) were identified as an ovel class of biorecognition elements for in vitro sensing,w hereby an indicator of the differential binding affinity between aT Fa nd its cognate DNAe xhibits dosedependent responsivity to an analyte.D escribed is am odular bead-based biosensor design that can be applied to such TF-DNA-analyte systems.D NA-functionalized beads enable efficient mixing and spatial separation, while TF-labeled semiconductor quantum dots serve as bright fluorescent indicators of the TF-DNAb ound (on bead) and unbound states.T he prototype sensor for derivatives of the antibiotic tetracycline exhibits nanomolar sensitivity with visual detection of bead fluorescence.F acile changes to the sensor enable sensor response tuning without necessitating changes to the biomolecular affinities.Assay components self-assemble,and readout by eye or digital camera is possible within 5minutes of analyte addition, making sensor use facile,rapid, and instrument-free.

Förster resonance energy transfer (FRET) is a widely used and an ideal transduction modality for fluorescent based biosensors as it offers high signal to noise with a visibly detectable signal....

Progesterone monitoring is an essential component of in vitro fertilization treatments and reproductive management of dairy cows. Gold-standard biosensors for progesterone monitoring rely on antibodies, which are expensive and difficult to procure. We have developed an alternative transcription factor-based sensor that is superior to conventional progesterone biosensors. Here, we incorporate this transcription factor-based progesterone sensor into an affordable, portable paperfluidic format to facilitate widespread implementation of progesterone monitoring at the point of care. Oligonucleotides labeled with a fluorescent dye are immobilized onto nitrocellulose via a biotin–streptavidin interaction. In the absence of progesterone, these oligonucleotides form a complex with a transcription factor that is fluorescently labeled with tdTomato. In the presence of progesterone, the fluorescent transcription factor unbinds from the immobilized DNA, resulting in a decrease in tdTomato fluorescence. The limit of detection of our system is 27 nm, which is a clinically relevant level of progesterone. We demonstrate that transcription factor-based sensors can be incorporated into paperfluidic devices, thereby making them accessible to a broader population due to the portability and affordability of paper-based devices.