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1.
Chem Sci ; 13(22): 6715-6731, 2022 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-35756504

RESUMO

Förster resonance energy transfer (FRET) is a widely used and ideal transduction modality for fluorescent based biosensors as it offers high signal to noise with a visibly detectable signal. While intense efforts are ongoing to improve the limit of detection and dynamic range of biosensors based on biomolecule optimization, the selection of and relative location of the dye remains understudied. Herein, we describe a combined experimental and computational study to systematically compare the nature of the dye, i.e., organic fluorophore (Cy5 or Texas Red) vs. inorganic nanoparticle (QD), and the position of the FRET donor or acceptor on the biomolecular components. Using a recently discovered transcription factor (TF)-deoxyribonucleic acid (DNA) biosensor for progesterone, we examine four different biosensor configurations and report the quantum yield, lifetime, FRET efficiency, IC50, and limit of detection. Fitting the computational models to the empirical data identifies key molecular parameters driving sensor performance in each biosensor configuration. Finally, we provide a set of design parameters to enable one to select the fluorophore system for future intermolecular biosensors using FRET-based conformational regulation in in vitro assays and new diagnostic devices.

2.
ACS Omega ; 7(7): 5804-5808, 2022 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-35224340

RESUMO

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.

3.
Angew Chem Int Ed Engl ; 59(48): 21597-21602, 2020 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-32945589

RESUMO

Recently, allosteric transcription factors (TFs) were identified as a novel class of biorecognition elements for in vitro sensing, whereby an indicator of the differential binding affinity between a TF and its cognate DNA exhibits dose-dependent responsivity to an analyte. Described is a modular bead-based biosensor design that can be applied to such TF-DNA-analyte systems. DNA-functionalized beads enable efficient mixing and spatial separation, while TF-labeled semiconductor quantum dots serve as bright fluorescent indicators of the TF-DNA bound (on bead) and unbound states. The prototype sensor for derivatives of the antibiotic tetracycline exhibits nanomolar sensitivity with visual detection of bead fluorescence. Facile 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 5 minutes of analyte addition, making sensor use facile, rapid, and instrument-free.


Assuntos
Antibacterianos/análise , Técnicas Biossensoriais , Telefone Celular , Corantes Fluorescentes/química , Tetraciclina/análise , Fatores de Transcrição/química , DNA/química , Pontos Quânticos/química , Semicondutores
4.
ACS Appl Mater Interfaces ; 12(39): 43513-43521, 2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-32893612

RESUMO

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 Förster 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.


Assuntos
DNA/química , Hidrogéis/química , Progesterona/análise , Pontos Quânticos/química , Fatores de Transcrição/química , Estrutura Molecular , Tamanho da Partícula , Propriedades de Superfície
5.
Adv Healthc Mater ; 9(17): e2000403, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32691962

RESUMO

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.


Assuntos
Técnicas Biossensoriais , Ácidos Nucleicos , Pontos Quânticos , Transferência Ressonante de Energia de Fluorescência , Fatores de Transcrição
6.
Small ; 16(17): e1907522, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32249506

RESUMO

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.


Assuntos
Técnicas Biossensoriais , DNA , Transferência Ressonante de Energia de Fluorescência , Fatores de Transcrição , Técnicas Biossensoriais/instrumentação , DNA/metabolismo , Corantes Fluorescentes , Pontos Quânticos , Fatores de Transcrição/metabolismo
7.
Nat Commun ; 11(1): 1276, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152281

RESUMO

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.


Assuntos
Actinobacteria/metabolismo , Técnicas Biossensoriais , Progesterona/metabolismo , Sequência de Bases , Transferência Ressonante de Energia de Fluorescência , Testes Imediatos , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo
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