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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 104 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., Gold leaf electrodes were fabricated and functionalized with methylene blue DNA. These electrodes were used to detect target-activated Cas12a endonuclease activity.

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.

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.

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.

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.

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.

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.

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