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We report a combined experimental and computational study to systematically compare the nature of the dye, i.e., organic fluorophore vs. inorganic nanoparticle, and the position of the FRET donor or acceptor on the biosensor performances.

Loop-mediated amplification (LAMP) is an isothermal amplification technique favored in diagnostics and point-of-care work due to its high sensitivity and ability to run in isothermal conditions. In addition, a visual readout by lateral flow strips (LFS) can be used in conjunction with LAMP, making the assay accessible at the point-of-care. However, the amplicons resulting from a LAMP reaction varied in length and shape, making them undiscernible on a double-stranded DNA intercalating dye stained gel. Standard characterization techniques also do not identify which amplicons specifically bind to the LFS, which generate the visual readout. We aimed to standardize our characterization of LAMP products during assay development by using fluorescein amidite (FAM) and biotin-tagged loop forward and backward primers during assay development. A pvuII restriction enzyme digest is applied to the LAMP products. FAM-tagged bands are directly correlated with the LFS visual readout. We applied this assay development workflow for an HPV 16 assay using both plasmid DNA and clinical samples to demonstrate proof of concept for generalized assay development work. Electronic supplementary material The online version of this article (10.1007/s00216-020-02702-9) contains supplementary material, which is available to authorized users.

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., Bacteria represent an unexploited reservoir of biosensing proteins. Here the authors use genomic screens and functional assays to isolate a progesterone sensing allosteric transcription factor and use a FRET-based method to develop an optical progesterone sensor.

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

In 2019, the first cases of SARS-CoV-2 were detected in Wuhan, China, and by early 2020 the cases were identified in the United States. SARS-CoV-2 infections increased in the US causing many states to implement stay-at-home orders and additional safety precautions to mitigate potential outbreaks. As policies changed throughout the pandemic and restrictions lifted, there was an increase in demand for Covid-19 testing which was costly, difficult to obtain, or had long turn-around times. Some academic institutions, including Boston University, created an on-campus Covid-19 screening protocol as part of planning for the safe return of students, faculty, and staff to campus with the option for in-person classes. At BU, we stood up an automated high-throughput clinical testing lab with the capacity to run 45,000 individual tests weekly by fall of 2020, with a purpose-built clinical testing laboratory, a multiplexed RT-PCR test, robotic instrumentation, and trained CLIA certified staff. There were challenges to overcome, including the supply chain issues for PPE testing materials, and equipment that were in high demand. The Boston University Clinical Testing Laboratory was operational at the start of the fall 2020 academic year. The lab performed over 1 million SARS-CoV-2 RT-PCR tests during the 2020-2021 academic year.

Monitoring the adherence of patients taking highly active antiretroviral therapy (HAART) is a key step in treating an HIV infection, especially in resource-limited settings in the developing world. For most regimens, when patients are not at least 95% adherent to their drug schedule, there is a loss of effectiveness in treatment resulting in increases in health care costs, increases in the rate of transmission, and reduction of positive patient outcomes. Currently, subjective methods such as pill counting, electronic drug monitoring, and patient self-reporting are the only ways clinicians can track adherence and intervene in cases of noncompliance. We address this issue by developing a dipstick-based point-of-care azide-alkyne click chemistry assay with colorimetric read-out that directly tests for the presence of one common HAART drug in patient urine. An alkyne-modified dextran was synthesized and characterized by NMR and then used to colorimetrically report the presence of azidothymidine, an azide-containing HAART drug, in urine samples. The assay is specific to azide-containing molecules that are not naturally present in the urine and is sensitive to physiologically relevant urine concentrations as low as 750 uM. This point-of-care device is a strong alternative in resource-limited settings over current direct measurement techniques that are expensive and require trained users such as HPLC.

Nucleic acid amplification tests (NAATs), which amplify and detect pathogen nucleic acids, are vital methods to diagnose diseases, particularly in cases where patients exhibit low levels of infection. For many blood-borne pathogens such as HIV or Plasmodium, it is necessary to first extract pathogen RNA or DNA from patient blood prior to analysis with NAATs. Traditional nucleic acid extraction methods are expensive, resource-intensive and are often difficult to deploy to resource-limited areas where many blood-borne infections are widespread. Here, we describe a portable, paper-and-plastic device for instrument-free nucleic acid extraction from whole blood, which we call SNAPflex, that builds upon our previous work extracting RNA in a 2D platform from nasopharyngeal swabs. We demonstrated improved extraction of HIV RNA from simulated patient samples compared to traditional extraction methods and long-term stability of extracted RNA without the need for cold storage. We further demonstrated successful extraction and recovery of Plasmodium falciparum DNA from simulated patient samples with superior recovery compared to existing extraction methods. The SNAPflex device extracts and purifies DNA and RNA from whole blood which can be amplified with traditional NAATs, and was designed to easily manufacture and integrate into existing health systems.

International audience; An alternative molecular recognition approach was developed for sensing small molecule analytes using the differential binding of an allosteric transcription factor (TF, specifically TetR) to its cognate DNA as the molecular recognition element coupled with fluorescent resonance energy transfer (FRET) to yield an internally calibrated optical signal transduction mechanism. Sensors were evaluated comprising Cy5-modified DNA (FRET acceptor) with either a tdTomato-TetR fusion protein (FP-TF) or quantum dot-TetR conjugate (QD-TF) as the FRET donor by measuring the ratio of acceptor and donor fluorescence intensities (FA/FD) with titrations of a derivative of the antibiotic tetracycline, anhydrous tetracycline (aTc). A proof-of-concept FRET-based biosensor was successfully demonstrated through the modulation of FA/FD signal intensities based on varying analyte concentrations. Sensor design parameters affecting overall signal-to-noise ratio and sensitivity of the sensors are also identified.

The glucose biosensor, built upon the redox enzyme glucose oxidase, is the most commercially successful and studied enzymatic biosensor. However, the lack of available and functionally validated enzymes is prohibiting the development of redox-based sensors for other important analytes. Herein, we present the development and assessment of an electrochemical nicotine biosensor, using genomic screening to identify the gene for a known nicotine catabolizing redox enzyme. The resulting nicotine biosensor demonstrated a specific, sensitive, and stable operational profile with a limit of detection of 27 μM over the range of 0-200 μM. This range is well within the physiological concentrations of nicotine present in smoker urine. Specificity and cross-reactivity were measured against structurally similar compounds to nicotine as well as to known physiological by-products. Our results highlight that this novel enzymatic electrochemical nicotine biosensor possesses operational capabilities for monitoring of nicotine in physiologically relevant conditions. The screening methodology can be generalized for the discovery of enzymes for novel sensor development.

Proper management of an HIV infection requires that a patient be at least 80–95% adherent to a prescribed drug regimen to avoid poor health outcomes and the development of drug-resistant HIV strains. Clinicians generally monitor adherence habits indirectly through patient self-reporting, pill counting, and electronic drug monitoring. While direct measurement of patient samples like urine for monitoring drug levels is possible, it requires specialized equipment and training that is not readily available in resource-limited settings where the need is greatest. In this work we report the development of an antibody that binds to tenofovir (TFV), a key small molecule drug for both the treatment and prevention of HIV, and a competitive lateral flow assay that uses that antibody to monitor urine samples for the presence of the drug. TFV was conjugated to an immunogenic protein and injected into rabbits to raise polyclonal antibodies sensitive to the drug. The antibodies were verified for TFV-sensitivity by immunoprecipitation and HPLC. A gold nanoparticle-based competitive assay was developed to detect the presence of TFV in urine samples with a sensitivity of 1 μg mL−1. This TFV assay could be deployed as a point-of-care device for adherence monitoring in resource-limited settings as a low-cost, accurate, and speedy alternative to current methods to better inform changes in treatment.

DNA extraction from clinical samples is commonly achieved with a silica solid phase extraction column in the presence of a chaotrope. Versions of these protocols have been adapted for point of care (POC) diagnostic devices in miniaturized platforms, but commercial kits require a high amount of input DNA. Thus, when the input clinical sample contains less than 1 μg of total DNA, the target-specific DNA recovery from most of these protocols is low without supplementing the sample with exogenous carrier DNA. In fact, many clinical samples used in the development of POC diagnostics often exhibit target DNA concentrations as low as 3 ng/mL. With the broader goal of improving the yield and efficiency of nucleic acid-based POC devices for dilute samples, we investigated both DNA adsorption and recovery from silica particles by using 1 pg- 1 μg of DNA with a set of adsorption and elution buffers ranging in pH and chaotropic presence. In terms of adsorption, we found that low pH and the presence of chaotropic guanidinium thiocyanate (GuSCN) enhanced DNA-silica adsorption. When eluting with a standard low-salt, high-pH buffer, > 70% of DNA was unrecoverable, except when DNA was initially adsorbed with 5 M GuSCN at pH 5.2. Unrecovered DNA was either not initially adsorbed or irreversibly bound on the silica surface. Recovery was improved when eluting with 95°C formamide and 1 M NaOH, which suggested that DNA-silica-chaotrope interactions are dominated by hydrophobic interactions and hydrogen bonding. While heated formamide and NaOH are non-ideal elution buffers for practical POC devices, the salient results are important for engineering a set of optimized reagents that could maximize nucleic acid recovery from a microfluidic DNA-silica-chaotrope system.

Herein we show the development of a minimally instrumented paper-based molecular diagnostic for point of care detection of sexually transmitted infections caused by Chlamydia trachomatis. This new diagnostic platform incorporates cell lysis, isothermal nucleic acid amplification, and lateral flow visual detection using only pressure and heat sources, eliminating the need for expensive laboratory equipment. This paper-based test can be performed in less than one hour and has a clinically relevant limit of detection that is 100× more sensitive than current rapid immunoassays used for chlamydia diagnosis.

Monitoring the adherence of patients taking highly-active anti-retroviral therapy (HAART) and Pre-Exposure Prophylaxis (PrEP) is a key step in treating an HIV infection. Since current monitor methods are non-direct there is a need for assays that directly verify the presence of active drug in the patient. In this work we modified an existing assay for the detection of cytosine to sense the presence of lamivudine (3TC) in spiked urine and emtricitabine (FTC) in water. 3TC and FTC is reacted with bromine and barium hydroxide to generate a colored precipitate in less than an hour indicating the presence of active drug. The current detection limit of the 3TC assay is 870uM in water and 2mM in urine. This assay can be applied in clinics to monitor patient adherence without the need for expensive equipment such as HPLC.

The 2009 Influenza A (H1N1) pandemic disproportionately affected the developing world and highlighted the key inadequacies of traditional diagnostic methods that make them unsuitable for use in resource-limited settings, from expensive equipment and infrastructure requirements to unacceptably long turnaround times. While rapid immunoassay diagnostic tests were much less costly and more context-appropriate, they suffered from drastically low sensitivities and high false negative rates. An accurate, sensitive, and specific molecular diagnostic that is also rapid, low-cost, and independent of laboratory infrastructure is needed for effective point-of-care detection and epidemiological control in these developing regions. We developed a paper-based assay that allows for the extraction and purification of RNA directly from human clinical nasopharyngeal specimens through a poly(ether sulfone) paper matrix, H1N1-specific in situ isothermal amplification directly within the same paper matrix, and immediate visual detection on lateral flow strips. The complete sample-to-answer assay can be performed at the point-of-care in just 45 min, without the need for expensive equipment or laboratory infrastructure, and it has a clinically relevant viral load detection limit of 10(6) copies/mL, offering a 10-fold improvement over current rapid immunoassays.

Herein we show the development of a minimally instrumented paper-based molecular diagnostic for point of care detection of sexually transmitted infections caused by Chlamydia trachomatis. This new diagnostic platform incorporates cell lysis, isothermal nucleic acid amplification, and lateral flow visual detection using only a pressure source and heat block, eliminating the need for expensive laboratory equipment. This paper-based test can be performed in less than one hour and has a clinically relevant limit of detection that is 100x more sensitive than current rapid immunoassays used for chlamydia diagnosis.

Wearable chemical sensor technologies enable the opportunity to continuously collect physiologically information on an individual’s health status. This is in contrast to traditional blood draws and subsequent analysis. Using glucose oxidase as a model enzyme we compare several fabrication methods and describe an optimized glucose monitoring sensor which works in open circuit potential with good selectivity and sensitivity over a glucose concentration range of 50 to 300 µM (Figure 1). The sensors maintain good sensitivity of more than 2000 nA/mM after two weeks. The current drifting is minimal and reproducible and device can be made. (Figure 1c). Key steps, procedural details, and findings will be discussed in this work. Figure 1. (a) The open circuit potential amperometric monitoring the liberated hydrogen peroxide after (a) 1 day of fabrication and (b) 15 days of fabrication; along with the calibration curves on inset. (c) Drifting and calibration curve obtained for sensors made from different batches. Figure 1

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

Often, modern diagnostic techniques require the isolation and purification of nucleic acids directly from patient samples such as blood or stool. Many diagnostic tests are being miniaturized onto micro-sized platforms and integrated into microfluidic devices due to the economies resulting from smaller sample and reagent volumes. Often, these devices perform sample preparation in series with the diagnostic tests. The sample preparation steps are vital in order to purify the desired genetic material from potential inhibitors that can interfere with the outcome of the test. There are various techniques used to selectively capture the nucleic acids while washing away potential contamination (proteins, enzymes, lipids, etc.). Two of the most common forms of selective capture are based on nucleic acid binding to silica surface or on the precipitation of nucleic acids with or without the presence of a carrier species. Each of these methods can be performed in liquid phase or in a solid support such as an extraction column. Here we discuss both methods and address microfluidic applications.

In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10(-2) pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health.

Many new and exciting portable HIV viral load testing technologies are emerging for use in global medicine. While the potential to provide fast, isothermal, and quantitative molecular diagnostic information to clinicians in the field will soon be a reality, many of these technologies lack a robust front end for sample clean up and nucleic acid preparation. Such a technology would enable many different downstream molecular assays. Here, we present a portable system for centrifuge-free room temperature nucleic acid extraction from small volumes of whole blood (70 mL), using only thermally stable reagents compatible with storage and transport in low resource settings. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis of simulated samples demonstrate a lower limit of detection of 1000 copies per mL, with the ability to detect differences in viral load across four orders of magnitude. The system can also be used to store extracted RNA on detachable cartridges for up to one week at ambient temperature, and can be operated using only hand generated air pressure.

Treating HIV in developing countries involves highly active antiretroviral therapies that require strict adherence by the patient. Current methods of self-reporting to monitor adherence to a drug regimen are less reliable than a drug test. This work introduces proof-of-concept of a new assay involving azide-alkyne cycloaddition to detect the presence of azidothymidine (AZT), a common antiretroviral therapy used in developing countries, in patient urine. Spotting experiments were performed on a paper-based platform with simple materials that selectively detects urine samples containing AZT and these results were confirmed by NMR and UV-Vis spectroscopy. The detection of the assay to AZT was found to be around 12mM. This assay can be translated into a point-of-care set-up that can be used by clinicians to directly monitor patient adherence while increasing efficiency and reducing the costs of health care for HIV patients.

The ability to increase the concentration of target analytes in a fixed sample volume can potentially lower the limit of detection for many biosensing techniques, and thus is key in sample preparation for infectious disease diagnosis. Concentration by evaporation is an effective method to achieve target enrichment. However, concentrating human samples, including blood and plasma, by evaporation-based methods is made challenging by high concentrations of proteins and electrolytes. Dehydration of the proteins causes the sample to turn into a gel, hindering further analysis. At the same time, decreasing the volume increases the overall concentration of electrolytes, causing bacterial or viral particle lysis, and making them more difficult to detect in affinity-based biosensors. Thus, we fabricated a microfluidic chip that incorporates both dialysis and concentration in a single design. The chip dialyzes the proteins from the plasma, while maintaining an appropriate concentration of electrolytes and concentrating the sample targets. The process to concentrate plasma or serum samples by a factor of 10 takes less than 30 minutes. As a proof-of-concept, we demonstrated the chip using a defective Human Immunodeficiency Virus (HIV). To distinguish patients on antiretroviral therapy who are failing therapy from those who are not, a diagnostic must be able to detect HIV in plasma down to at least 1000 particles per milliliter. For a number of technical reasons, it is difficult to get on-chip PCR reactions to reach this level of sensitivity, so concentration of HIV from lower viral load samples has the potential to improve the sensitivity of many types of molecular point-of-care viral load tests.

Correction for ‘Paper-based molecular diagnostic for Chlamydia trachomatis’ by Jacqueline C. Linnes et al., RSC Adv., 2014, 4, 42245–42251.

a) The current gold-standard of care in the management of patients with thyroid nodules is ultrasound-guided fine needle aspiration biopsy (FNAB) followed by microscopic examination of cell morphology by a trained cytopathologist. Due to the lack of distinguishing morphology, 10-25% of FNAs are termed “indeterminate” and required surgery. However, only 35% of them are found to have cancer. There is a need for a more accurate and minimally invasive cancer diagnostic technology. Our objective is to develop an inexpensive, point-of-care molecular diagnostic platform to isolate and detect thyroid specific proteins to enable real-world use of biomarkers to inform patient care. b) In this study, we engineer a miniature ion exchange column within a plastic (cyclic olefin polymer), disposable lab-on-a-chip platform for sample preparation and protein purification using microfluidic channels with a specialized porous polymer monolith (PPM)-based resin bed. We capitalized on the finding that cancer cells exhibit differential protein expressional patterns compared to normal thyroid cells by targeting thyroid transcription factor (TTF-1), a thyroid specific enhancer binding protein, as a biomarker for the diagnosis of thyroid cancer. The ability to capture and release TTF-1 from the papillary thyroid cancer (PTC) cell line was evaluated via western blot. We also tested various types of thyroid samples obtained from malignant and benign human thyroid nodules. c) We compared the efficiency of a small-scale protein prep using a commercial gravity column versus our lab-on-a-chip column. Our method showed TTF-1 protein was detectable from the lysate of 5x104 cultured BCPAP cancer cell line. In contrast, it was not detectable when purified by gravity ion-exchange column. We next showed that one can maximize the concentration of protein eluted from as fewer cells as possible. We found that by titrating the elution volume per fraction down to 10 µl, the final concentration of eluted protein can be increased, and hence increase the downstream LOD to 104 cells. Next, we evaluated our extraction and purification system by using 10 mg patient thyroid tissue samples. We tested 11 human thyroid specimens for TTF-1 protein capture. They were all found to be positive which correlated with the official histopathological findings. All tissue extracts had measurable levels of TTF-1 protein and the levels of the TTF-1 were variable in the tissue specimens. We also tested one negative thyroid specimen. No TTF-1 protein was found. d) Our microfluidic protein extraction and purification system is a platform technology that may allow for optimal use of low-volume sample preparation such as we see in thyroid biopsies. This manner, when combined with an on-chip ELISA assay, would be a simple, cost effective test that gives the doctor all the needed information quickly in a single test. When mature, the device can be applied to other type of cancer based on tailored assays for specific biomarkers. Citation Format: Shichu Huang, Siddhartha Sharma, Lena Liu, Andy Fan, Catherine Klapperich, Jennifer Rosen. Microfluidic platform for a protein-based thyroid cancer diagnostics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3494. doi:10.1158/1538-7445.AM2014-3494