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.

Sexually transmitted infections, including the human immunodeficiency virus (HIV) and the human papillomavirus (HPV), disproportionally impact those in low-resource settings. Early diagnosis is essential for managing HIV. Similarly, HPV causes nearly all cases of cervical cancer, the majority (90%) of which occur in low-resource settings. Importantly, infection with HPV is six times more likely to progress to cervical cancer in women who are HIV-positive. An inexpensive, adaptable point-of-care test for viral infections would make screening for these viruses more accessible to a broader set of the population. Here, we report a novel, cost-effective electrochemical platform using gold leaf electrodes to detect clinically relevant viral loads. We have combined this platform with loop-mediated isothermal amplification and a CRISPR-based recognition assay to detect HPV. Lower limits of detection were demonstrated down to 10 4 total copies of input nucleic acids, which is a clinically relevant viral load for HPV DNA. Further, proof-of-concept experiments with cervical swab samples, extracted using standard extraction protocols, demonstrated that the strategy is extendable to complex human samples. This adaptable technology could be applied to detect any viral infection rapidly and cost-effectively.

Electrochemical biosensors are promising technologies for detection and monitoring in low-resource settings due to their potential for easy use and low-cost instrumentation. Disposable gold screen-printed electrodes (SPEs) are popular substrates for these biosensors, but necessary dopants in the ink used for their production can interfere with biosensor function and contribute to the heterogeneity of these electrodes. We recently reported an alternative disposable gold electrode made from gold leaf generated using low-cost, equipment-free fabrication. We have directly compared the surface topology, biorecognition element deposition, and functional performance of three disposable gold electrodes: our gold leaf electrodes and two commercial SPEs. Our leaf electrodes significantly outperformed the SPEs for reproducible and effective biosensing in a DNase I assay and are nearly an order of magnitude less expensive than the SPEs. Therefore, these electrodes are promising for further development as point-of-care diagnostics, especially in low-resource settings.

Conspectus Disease prevalence is highest in low-resource settings (LRS) due to the lack of funds, infrastructure, and personnel required to carry out laboratory-based molecular tests. In high-resource settings, gold-standard molecular tests for diseases consist of nucleic acid amplification tests (NAATs) due to their excellent sensitivity and specificity. These tests require the extraction, amplification, and detection of nucleic acids from clinical samples. In high-resource settings, all three of these steps require highly specialized, costly, and onerous equipment that cannot be used in LRS. Nucleic acid extraction involves multiple centrifugation steps. Amplification consists of the polymerase chain reaction (PCR), which requires thermal cyclers. The detection of amplified DNA is typically done with specialized thermal cyclers that are capable of fluorescence detection. Traditional methods used to extract, amplify, and detect nucleic acids cannot be used outside of a laboratory in LRS. Thus, there is a need for affordable point-of-care devices to ease the high burden of disease in LRS. The past decade of work on paper-based fluidic devices has resulted in the invention of many paper-based biosensors for disease detection as well as isothermal amplification techniques that replace PCR. However, a challenge still remains in detecting pathogenic biomarkers from complex human samples without specialized laboratory equipment. Our research has focused on the development of affordable technologies to extract and detect nucleic acids in clinical samples with minimal equipment. Here we describe methods for the paper-based extraction, amplification, and detection of nucleic acids. This Account provides an overview of our latest technologies developed to detect an array of diseases in low-resource settings. We focus on detecting nucleic acids of H1N1, human papillomavirus (HPV), Neisseria gonorrheae (NG), Chlamydia trachomatis (CT), Trichomonas vaginalis (TV), and malaria from a variety of clinical sample types. H1N1 RNA was extracted from nasopharyngeal swabs; HPV, NG, and CT DNA were extracted from either cervical, urethral, or vaginal swabs; TV DNA was extracted from urine; and malaria DNA was extracted from whole blood. Different sample types necessitate different nucleic extraction protocols; we provide guidelines for assay design based on the clinical sample type used. We compare the pros and cons of different isothermal amplification techniques, namely, helicase-dependent amplification (HDA), loop-mediated isothermal amplification (LAMP), and a novel isothermal amplification technique that we developed: isothermal-identical multirepeat sequences (iso-IMRS). Finally, we compare various detection mechanisms, including lateral-flow and electrochemical readouts. Electrochemical readouts frequently employ gold electrodes due to strong gold–thiol coupling. However, the high cost of gold precludes their use in LRS. We discuss our development of novel gold leaf electrodes that can be made without specialized equipment for...

Conventional gold electrode fabrication is too costly and laborious for implementation in low-resource settings (LRS). We review affordable, simple alternative fabrication methods, highlighting gold leaf electrodes, for LRS applications.

Therapeutic monitoring of neurotransmitters (NTs) and psychiatric medications is essential for the diagnosis and treatment of mental illness. However, in-vivo monitoring of NTs in humans as well as continuous physiological monitoring of psychiatrics have yet to be realized. In pursuit of this goal, there has been a plethora of work to develop electrochemical sensors for both in-vivo NT monitoring as well as in-vitro detection of psychiatric medications. We review these sensors here while discussing next steps needed to achieve concurrent, continuous physiological monitoring of NTs and psychiatric medications as part of a closed-loop feedback system that guides medication administration.

Electrochemical sensors to measure biomarkers from complex samples are a tried and tested technology with large untapped potential for addressing important public health needs.

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.