15 results on '"Andy Fan"'
Search Results
2. Electrochemical Strategy for Low-Cost Viral Detection
- Author
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Marjon Zamani, James M. Robson, Andy Fan, Michael S. Bono, Ariel L. Furst, and Catherine M. Klapperich
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Chemistry ,QD1-999 - Published
- 2021
- Full Text
- View/download PDF
3. Method for the elucidation of LAMP products captured on lateral flow strips in a point of care test for HPV 16
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Lena Landaverde, Andy Fan, Catherine M. Klapperich, Winnie S. Wong, and Gabriela Hernandez
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Materials science ,HPV 16 ,genetic structures ,Point-of-Care Systems ,Loop-mediated isothermal amplification ,02 engineering and technology ,STRIPS ,Proof of Concept Study ,Sensitivity and Specificity ,01 natural sciences ,Biochemistry ,Analytical Chemistry ,law.invention ,chemistry.chemical_compound ,Plasmid dna ,law ,Humans ,Fluorescent ,Nucleic acid amplification ,Paper in Forefront ,Human papillomavirus 16 ,Chromatography ,Assay development ,010401 analytical chemistry ,Lateral flow strip ,DNA ,Amplicon ,Loop-mediated amplification ,021001 nanoscience & nanotechnology ,Fluorescence ,eye diseases ,Point of care ,0104 chemical sciences ,Fluorescein amidite ,chemistry ,Restriction digest ,0210 nano-technology ,Nucleic Acid Amplification Techniques - Abstract
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.
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- 2020
4. A progesterone biosensor derived from microbial screening
- Author
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James E. Galagan, Mingfu Chen, Patricia Aquino, Thuy T. Nguyen, Xiaoman Zhang, Margaret Chern, Catherine M. Klapperich, Mario Cabodi, R C. Baer, Sébastien Lecommandoux, Marjon Zamani, Andy Fan, Mark W. Grinstaff, Uros Kuzmanovic, Allison M. Dennis, Chloé Grazon, Laboratoire de Chimie des Polymères Organiques (LCPO), Université de Bordeaux (UB)-Ecole Nationale Supérieure de Chimie, de Biologie et de Physique (ENSCBP)-Institut Polytechnique de Bordeaux-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Team 3 LCPO : Polymer Self-Assembly & Life Sciences, Université de Bordeaux (UB)-Ecole Nationale Supérieure de Chimie, de Biologie et de Physique (ENSCBP)-Institut Polytechnique de Bordeaux-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux (UB)-Ecole Nationale Supérieure de Chimie, de Biologie et de Physique (ENSCBP)-Institut Polytechnique de Bordeaux-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Boston University [Boston] (BU), National Emerging Infectious Diseases Laboratories, Department of Biomedical Engineering [Boston], Division of Materials Science and Engineering, DARPA (W911NF-16-C-0044), NIH U54EB015403, NIH CTSI 1KL2TR001411, NIH S10 OD016326, European Project: MSCA-IF-2016 749973,SENSHOR, Centre National de la Recherche Scientifique (CNRS)-Institut Polytechnique de Bordeaux-Ecole Nationale Supérieure de Chimie, de Biologie et de Physique (ENSCBP)-Université de Bordeaux (UB)-Institut de Chimie du CNRS (INC), and Centre National de la Recherche Scientifique (CNRS)-Institut Polytechnique de Bordeaux-Ecole Nationale Supérieure de Chimie, de Biologie et de Physique (ENSCBP)-Université de Bordeaux (UB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Polytechnique de Bordeaux-Ecole Nationale Supérieure de Chimie, de Biologie et de Physique (ENSCBP)-Université de Bordeaux (UB)-Institut de Chimie du CNRS (INC)
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0301 basic medicine ,Analyte ,[SDV.BIO]Life Sciences [q-bio]/Biotechnology ,Science ,General Physics and Astronomy ,02 engineering and technology ,Computational biology ,Biosensing Techniques ,General Biochemistry, Genetics and Molecular Biology ,Article ,Bioassays ,03 medical and health sciences ,Synthetic biology ,Fluorescence Resonance Energy Transfer ,Base sequence ,[SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials ,lcsh:Science ,Progesterone ,Multidisciplinary ,Base Sequence ,Chemistry ,Extramural ,Reproducibility of Results ,General Chemistry ,Genomics ,[SDV.MHEP.EM]Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism ,021001 nanoscience & nanotechnology ,Actinobacteria ,030104 developmental biology ,Förster resonance energy transfer ,Biosensors ,[CHIM.POLY]Chemical Sciences/Polymers ,Artificial urine ,Point-of-Care Testing ,Progesterone metabolism ,lcsh:Q ,0210 nano-technology ,Biosensor ,Biomedical engineering ,Transcription Factors - Abstract
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.
- Published
- 2020
5. Colorimetric Detection of Azidothymidine Using an Alkyne-Modified Dextran Substrate
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Andy Fan, George W. Pratt, and Catherine M. Klapperich
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Drug ,medicine.medical_specialty ,business.industry ,media_common.quotation_subject ,Biomedical Engineering ,Human immunodeficiency virus (HIV) ,Dipstick ,Pharmacology ,medicine.disease_cause ,Positive patient ,Antiretroviral therapy ,Biomaterials ,chemistry.chemical_compound ,Dextran ,chemistry ,Internal medicine ,medicine ,In patient ,business ,media_common - Abstract
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.
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- 2021
6. SNAPflex: a paper-and-plastic device for instrument-free RNA and DNA extraction from whole blood
- Author
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Nikunja Kolluri, Andy Fan, Catherine M. Klapperich, Anna Young, Nikolas Albarran, Alex Olson, Jose Gomez-Marquez, and Manish Sagar
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Biomedical Engineering ,Cold storage ,Bioengineering ,02 engineering and technology ,Computational biology ,Biology ,01 natural sciences ,Biochemistry ,chemistry.chemical_compound ,Humans ,Nucleic Acid Amplification Tests ,Malaria, Falciparum ,Whole blood ,010401 analytical chemistry ,Extraction (chemistry) ,RNA ,DNA ,General Chemistry ,021001 nanoscience & nanotechnology ,DNA extraction ,0104 chemical sciences ,chemistry ,Nucleic acid ,RNA extraction ,0210 nano-technology ,Nucleic Acid Amplification Techniques ,Plastics - Abstract
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.
- Published
- 2020
7. QD-FRET-based biosensing of small molecule analytes using transcription factor-DNA binding
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Chloé Grazon, Andy Fan, Thuy T. Nguyen, Margaret Chern, R C. Baer, Allison M. Dennis, James E. Galagan, Boston University [Boston] (BU), Department of Biomedical Engineering [Boston], National Emerging Infectious Diseases Laboratories (NEIDL), Department of Chemistry, Boston University, Laboratoire de Chimie des Polymères Organiques (LCPO), Université de Bordeaux (UB)-Ecole Nationale Supérieure de Chimie, de Biologie et de Physique (ENSCBP)-Institut Polytechnique de Bordeaux-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Team 3 LCPO : Polymer Self-Assembly & Life Sciences, Université de Bordeaux (UB)-Ecole Nationale Supérieure de Chimie, de Biologie et de Physique (ENSCBP)-Institut Polytechnique de Bordeaux-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux (UB)-Ecole Nationale Supérieure de Chimie, de Biologie et de Physique (ENSCBP)-Institut Polytechnique de Bordeaux-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), European Project: MSCA-IF-2016 749973,SENSHOR, Centre National de la Recherche Scientifique (CNRS)-Institut Polytechnique de Bordeaux-Ecole Nationale Supérieure de Chimie, de Biologie et de Physique (ENSCBP)-Université de Bordeaux (UB)-Institut de Chimie du CNRS (INC), and Centre National de la Recherche Scientifique (CNRS)-Institut Polytechnique de Bordeaux-Ecole Nationale Supérieure de Chimie, de Biologie et de Physique (ENSCBP)-Université de Bordeaux (UB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Polytechnique de Bordeaux-Ecole Nationale Supérieure de Chimie, de Biologie et de Physique (ENSCBP)-Université de Bordeaux (UB)-Institut de Chimie du CNRS (INC)
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Analyte ,[CHIM.MATE]Chemical Sciences/Material chemistry ,02 engineering and technology ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,Fluorescence ,Small molecule ,0104 chemical sciences ,chemistry.chemical_compound ,[CHIM.POLY]Chemical Sciences/Polymers ,Molecular recognition ,Förster resonance energy transfer ,chemistry ,Biophysics ,TetR ,0210 nano-technology ,[PHYS.COND.CM-SCM]Physics [physics]/Condensed Matter [cond-mat]/Soft Condensed Matter [cond-mat.soft] ,Biosensor ,DNA - Abstract
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.
- Published
- 2019
8. An Enzymatic Electrochemical Biosensor for Real-Time Detection of Physiologically Relevant Nicotine Concentrations
- Author
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Andy Fan, Uros Kuzmanovic, Margarita A. Tararina, Nicolas Shijie Shu, Catherine M. Klapperich, Anant Gupta, Mingfu Chen, Karen N. Allen, Mark W. Grinstaff, Prerana Sensharma, and James E. Galagan
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chemistry.chemical_classification ,Nicotine ,Enzyme ,Chromatography ,chemistry ,technology, industry, and agriculture ,medicine ,Electrochemical biosensor ,medicine.drug - Abstract
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.
- Published
- 2020
9. A Competitive Lateral Flow Assay for the Detection of Tenofovir
- Author
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Bissrat Melakeberhan, Andy Fan, George W. Pratt, and Catherine M. Klapperich
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0301 basic medicine ,Drug ,Tenofovir ,Anti-HIV Agents ,media_common.quotation_subject ,Human immunodeficiency virus (HIV) ,Urine ,Pharmacology ,Health outcomes ,medicine.disease_cause ,Biochemistry ,Article ,Antibodies ,Analytical Chemistry ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Environmental Chemistry ,Animals ,Humans ,Immunoprecipitation ,030212 general & internal medicine ,Drug regimen ,Spectroscopy ,Chromatography, High Pressure Liquid ,media_common ,biology ,Chemistry ,030104 developmental biology ,Adherence monitoring ,biology.protein ,Rabbits ,Antibody ,medicine.drug - Abstract
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.
- Published
- 2018
10. Low concentration DNA extraction and recovery using a silica solid phase
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Andy Fan, Constantinos Katevatis, and Catherine M. Klapperich
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0301 basic medicine ,Formamide ,lcsh:Medicine ,01 natural sciences ,Physical Chemistry ,Polymerase Chain Reaction ,Guanidinium thiocyanate ,chemistry.chemical_compound ,Limit of Detection ,Solid phase extraction ,lcsh:Science ,DNA extraction ,Multidisciplinary ,Formamides ,Chemistry ,Organic Compounds ,Hydrogen-Ion Concentration ,Silicon Dioxide ,Precipitation Techniques ,Chaotropic agent ,Separation Processes ,Physical Sciences ,Ethanol Precipitation ,Sorption ,Hydrophobic and Hydrophilic Interactions ,Research Article ,Research and Analysis Methods ,03 medical and health sciences ,Adsorption ,Extraction techniques ,Solid-Phase Extraction ,Ethanol precipitation ,Chromatography ,Miniaturization ,Chemical Bonding ,Ethanol ,Elution ,010401 analytical chemistry ,lcsh:R ,Organic Chemistry ,Chemical Compounds ,Organic Solvent Precipitation ,Hydrogen Bonding ,DNA ,Amides ,0104 chemical sciences ,030104 developmental biology ,Alcohols ,lcsh:Q - Abstract
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.
- Published
- 2017
11. Detection of lamivudine and emtricitabine using a modified pyrimidine assay
- Author
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George W. Pratt, Catherine M. Klapperich, and Andy Fan
- Subjects
Detection limit ,Drug ,Pyrimidine ,Chemistry ,media_common.quotation_subject ,Human immunodeficiency virus (HIV) ,Lamivudine ,Urine ,Pharmacology ,medicine.disease_cause ,Emtricitabine ,chemistry.chemical_compound ,medicine ,medicine.drug ,media_common - Abstract
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.
- Published
- 2016
12. Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens
- Author
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Nira R. Pollock, Courtney K. Ellenson, Catherine M. Klapperich, Jacqueline C. Linnes, Andy Fan, and Natalia M. Rodriguez
- Subjects
In situ ,Paper ,Diagnostic methods ,Chemistry ,Point-of-Care Systems ,Loop-mediated isothermal amplification ,Nanotechnology ,Influenza a ,Paper based ,Article ,Analytical Chemistry ,Reliability engineering ,Influenza A Virus, H1N1 Subtype ,Genetic Techniques ,Limit of Detection ,Flow detection ,Influenza, Human ,Humans ,RNA ,RNA extraction ,Developing regions - Abstract
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.
- Published
- 2015
13. Purification of DNA/RNA in a microfluidic device
- Author
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Samantha Byrnes, Andy Fan, and Catherine M. Klapperich
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DNA, Bacterial ,Chromatography ,Ultraviolet Rays ,Sample (material) ,Microfluidics ,Solid Phase Extraction ,RNA ,Chemical Fractionation ,Microfluidic Analytical Techniques ,Molecular biology ,Article ,chemistry.chemical_compound ,chemistry ,Reagent ,Nucleic acid ,Humans ,RNA, Viral ,Sample preparation ,Solid phase extraction ,Plastics ,DNA - Abstract
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.
- Published
- 2013
14. Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis
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Lei Zhao, Catherine M. Klapperich, Jaephil Do, Lisa I. Jepeal, Andy Fan, Shichu Huang, Satish K. Singh, and Madhumita Mahalanabis
- Subjects
Bacterial Diseases ,Diarrhea ,Clostridium Difficile ,Science ,Microfluidics ,Loop-mediated isothermal amplification ,Biomedical Engineering ,Bioengineering ,Gastroenterology and Hepatology ,Global Health ,01 natural sciences ,Microbiology ,Medical Devices ,03 medical and health sciences ,Engineering ,Humans ,Sample preparation ,Gastrointestinal Infections ,Solid phase extraction ,030304 developmental biology ,Detection limit ,0303 health sciences ,Multidisciplinary ,Chromatography ,Chemistry ,Clostridioides difficile ,010401 analytical chemistry ,Extraction (chemistry) ,Temperature ,Nucleic acid amplification technique ,DNA ,DNA extraction ,3. Good health ,0104 chemical sciences ,Infectious Diseases ,Medicine ,Nucleic Acid Amplification Techniques ,Research Article - Abstract
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.
- Published
- 2013
15. Sample concentration and purification for point-of-care diagnostics
- Author
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Mario Cabodi, Andy Fan, Catherine M. Klapperich, and Nga T. Ho
- Subjects
Flow injection analysis ,Detection limit ,Blood Specimen Collection ,Analyte ,Chromatography ,Lysis ,Chemistry ,Point-of-Care Systems ,Sample (material) ,Reproducibility of Results ,Blood Proteins ,Equipment Design ,Microfluidic Analytical Techniques ,Sensitivity and Specificity ,Blood proteins ,Article ,Equipment Failure Analysis ,Flow Injection Analysis ,Immunology ,Humans ,Sample preparation ,Viral load ,Blood Chemical Analysis - Abstract
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.
- Published
- 2012
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