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Showing 1 - 200 of 3120 Journals sorted alphabetically
A Practical Logic of Cognitive Systems     Full-text available via subscription   (Followers: 8)
AASRI Procedia     Open Access   (Followers: 15)
Academic Pediatrics     Hybrid Journal   (Followers: 26, SJR: 1.402, h-index: 51)
Academic Radiology     Hybrid Journal   (Followers: 22, SJR: 1.008, h-index: 75)
Accident Analysis & Prevention     Partially Free   (Followers: 90, SJR: 1.109, h-index: 94)
Accounting Forum     Hybrid Journal   (Followers: 25, SJR: 0.612, h-index: 27)
Accounting, Organizations and Society     Hybrid Journal   (Followers: 30, SJR: 2.515, h-index: 90)
Achievements in the Life Sciences     Open Access   (Followers: 4)
Acta Anaesthesiologica Taiwanica     Open Access   (Followers: 5, SJR: 0.338, h-index: 19)
Acta Astronautica     Hybrid Journal   (Followers: 381, SJR: 0.726, h-index: 43)
Acta Automatica Sinica     Full-text available via subscription   (Followers: 3)
Acta Biomaterialia     Hybrid Journal   (Followers: 26, SJR: 2.02, h-index: 104)
Acta Colombiana de Cuidado Intensivo     Full-text available via subscription   (Followers: 1)
Acta de Investigación Psicológica     Open Access   (Followers: 2)
Acta Ecologica Sinica     Open Access   (Followers: 8, SJR: 0.172, h-index: 29)
Acta Haematologica Polonica     Free   (Followers: 1, SJR: 0.123, h-index: 8)
Acta Histochemica     Hybrid Journal   (Followers: 3, SJR: 0.604, h-index: 38)
Acta Materialia     Hybrid Journal   (Followers: 239, SJR: 3.683, h-index: 202)
Acta Mathematica Scientia     Full-text available via subscription   (Followers: 5, SJR: 0.615, h-index: 21)
Acta Mechanica Solida Sinica     Full-text available via subscription   (Followers: 9, SJR: 0.442, h-index: 21)
Acta Oecologica     Hybrid Journal   (Followers: 10, SJR: 0.915, h-index: 53)
Acta Otorrinolaringologica (English Edition)     Full-text available via subscription   (Followers: 1)
Acta Otorrinolaringológica Española     Full-text available via subscription   (Followers: 3, SJR: 0.311, h-index: 16)
Acta Pharmaceutica Sinica B     Open Access   (Followers: 2)
Acta Poética     Open Access   (Followers: 4)
Acta Psychologica     Hybrid Journal   (Followers: 25, SJR: 1.365, h-index: 73)
Acta Sociológica     Open Access  
Acta Tropica     Hybrid Journal   (Followers: 6, SJR: 1.059, h-index: 77)
Acta Urológica Portuguesa     Open Access  
Actas Dermo-Sifiliograficas     Full-text available via subscription   (Followers: 4)
Actas Dermo-Sifiliográficas (English Edition)     Full-text available via subscription   (Followers: 3)
Actas Urológicas Españolas     Full-text available via subscription   (Followers: 4, SJR: 0.383, h-index: 19)
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Actualites Pharmaceutiques     Full-text available via subscription   (Followers: 5, SJR: 0.141, h-index: 3)
Actualites Pharmaceutiques Hospitalieres     Full-text available via subscription   (Followers: 4, SJR: 0.112, h-index: 2)
Acupuncture and Related Therapies     Hybrid Journal   (Followers: 5)
Acute Pain     Full-text available via subscription   (Followers: 13)
Ad Hoc Networks     Hybrid Journal   (Followers: 11, SJR: 0.967, h-index: 57)
Addictive Behaviors     Hybrid Journal   (Followers: 15, SJR: 1.514, h-index: 92)
Addictive Behaviors Reports     Open Access   (Followers: 7)
Additive Manufacturing     Hybrid Journal   (Followers: 7, SJR: 1.039, h-index: 5)
Additives for Polymers     Full-text available via subscription   (Followers: 22)
Advanced Cement Based Materials     Full-text available via subscription   (Followers: 3)
Advanced Drug Delivery Reviews     Hybrid Journal   (Followers: 142, SJR: 5.2, h-index: 222)
Advanced Engineering Informatics     Hybrid Journal   (Followers: 11, SJR: 1.265, h-index: 53)
Advanced Powder Technology     Hybrid Journal   (Followers: 17, SJR: 0.739, h-index: 33)
Advances in Accounting     Hybrid Journal   (Followers: 9, SJR: 0.299, h-index: 15)
Advances in Agronomy     Full-text available via subscription   (Followers: 15, SJR: 2.071, h-index: 82)
Advances in Anesthesia     Full-text available via subscription   (Followers: 27, SJR: 0.169, h-index: 4)
Advances in Antiviral Drug Design     Full-text available via subscription   (Followers: 4)
Advances in Applied Mathematics     Full-text available via subscription   (Followers: 9, SJR: 1.054, h-index: 35)
Advances in Applied Mechanics     Full-text available via subscription   (Followers: 12, SJR: 0.801, h-index: 26)
Advances in Applied Microbiology     Full-text available via subscription   (Followers: 23, SJR: 1.286, h-index: 49)
Advances In Atomic, Molecular, and Optical Physics     Full-text available via subscription   (Followers: 16, SJR: 3.31, h-index: 42)
Advances in Biological Regulation     Hybrid Journal   (Followers: 4, SJR: 2.277, h-index: 43)
Advances in Botanical Research     Full-text available via subscription   (Followers: 3, SJR: 0.619, h-index: 48)
Advances in Cancer Research     Full-text available via subscription   (Followers: 26, SJR: 2.215, h-index: 78)
Advances in Carbohydrate Chemistry and Biochemistry     Full-text available via subscription   (Followers: 9, SJR: 0.9, h-index: 30)
Advances in Catalysis     Full-text available via subscription   (Followers: 6, SJR: 2.139, h-index: 42)
Advances in Cell Aging and Gerontology     Full-text available via subscription   (Followers: 4)
Advances in Cellular and Molecular Biology of Membranes and Organelles     Full-text available via subscription   (Followers: 13)
Advances in Chemical Engineering     Full-text available via subscription   (Followers: 26, SJR: 0.183, h-index: 23)
Advances in Child Development and Behavior     Full-text available via subscription   (Followers: 10, SJR: 0.665, h-index: 29)
Advances in Chronic Kidney Disease     Full-text available via subscription   (Followers: 9, SJR: 1.268, h-index: 45)
Advances in Clinical Chemistry     Full-text available via subscription   (Followers: 29, SJR: 0.938, h-index: 33)
Advances in Colloid and Interface Science     Full-text available via subscription   (Followers: 18, SJR: 2.314, h-index: 130)
Advances in Computers     Full-text available via subscription   (Followers: 16, SJR: 0.223, h-index: 22)
Advances in Dermatology     Full-text available via subscription   (Followers: 12)
Advances in Developmental Biology     Full-text available via subscription   (Followers: 12)
Advances in Digestive Medicine     Open Access   (Followers: 7)
Advances in DNA Sequence-Specific Agents     Full-text available via subscription   (Followers: 6)
Advances in Drug Research     Full-text available via subscription   (Followers: 23)
Advances in Ecological Research     Full-text available via subscription   (Followers: 47, SJR: 3.25, h-index: 43)
Advances in Engineering Software     Hybrid Journal   (Followers: 27, SJR: 0.486, h-index: 10)
Advances in Experimental Biology     Full-text available via subscription   (Followers: 9)
Advances in Experimental Social Psychology     Full-text available via subscription   (Followers: 45, SJR: 5.465, h-index: 64)
Advances in Exploration Geophysics     Full-text available via subscription   (Followers: 3)
Advances in Food and Nutrition Research     Full-text available via subscription   (Followers: 52, SJR: 0.674, h-index: 38)
Advances in Fuel Cells     Full-text available via subscription   (Followers: 16)
Advances in Genetics     Full-text available via subscription   (Followers: 17, SJR: 2.558, h-index: 54)
Advances in Genome Biology     Full-text available via subscription   (Followers: 11)
Advances in Geophysics     Full-text available via subscription   (Followers: 6, SJR: 2.325, h-index: 20)
Advances in Heat Transfer     Full-text available via subscription   (Followers: 22, SJR: 0.906, h-index: 24)
Advances in Heterocyclic Chemistry     Full-text available via subscription   (Followers: 9, SJR: 0.497, h-index: 31)
Advances in Human Factors/Ergonomics     Full-text available via subscription   (Followers: 27)
Advances in Imaging and Electron Physics     Full-text available via subscription   (Followers: 2, SJR: 0.396, h-index: 27)
Advances in Immunology     Full-text available via subscription   (Followers: 36, SJR: 4.152, h-index: 85)
Advances in Inorganic Chemistry     Full-text available via subscription   (Followers: 9, SJR: 1.132, h-index: 42)
Advances in Insect Physiology     Full-text available via subscription   (Followers: 3, SJR: 1.274, h-index: 27)
Advances in Integrative Medicine     Hybrid Journal   (Followers: 6)
Advances in Intl. Accounting     Full-text available via subscription   (Followers: 4)
Advances in Life Course Research     Hybrid Journal   (Followers: 8, SJR: 0.764, h-index: 15)
Advances in Lipobiology     Full-text available via subscription   (Followers: 2)
Advances in Magnetic and Optical Resonance     Full-text available via subscription   (Followers: 10)
Advances in Marine Biology     Full-text available via subscription   (Followers: 16, SJR: 1.645, h-index: 45)
Advances in Mathematics     Full-text available via subscription   (Followers: 10, SJR: 3.261, h-index: 65)
Advances in Medical Sciences     Hybrid Journal   (Followers: 6, SJR: 0.489, h-index: 25)
Advances in Medicinal Chemistry     Full-text available via subscription   (Followers: 6)
Advances in Microbial Physiology     Full-text available via subscription   (Followers: 5, SJR: 1.44, h-index: 51)
Advances in Molecular and Cell Biology     Full-text available via subscription   (Followers: 23)
Advances in Molecular and Cellular Endocrinology     Full-text available via subscription   (Followers: 10)
Advances in Molecular Toxicology     Full-text available via subscription   (Followers: 9, SJR: 0.324, h-index: 8)
Advances in Nanoporous Materials     Full-text available via subscription   (Followers: 4)
Advances in Oncobiology     Full-text available via subscription   (Followers: 2)
Advances in Organ Biology     Full-text available via subscription   (Followers: 2)
Advances in Organometallic Chemistry     Full-text available via subscription   (Followers: 15, SJR: 2.885, h-index: 45)
Advances in Parallel Computing     Full-text available via subscription   (Followers: 7, SJR: 0.148, h-index: 11)
Advances in Parasitology     Full-text available via subscription   (Followers: 7, SJR: 2.37, h-index: 73)
Advances in Pediatrics     Full-text available via subscription   (Followers: 24, SJR: 0.4, h-index: 28)
Advances in Pharmaceutical Sciences     Full-text available via subscription   (Followers: 13)
Advances in Pharmacology     Full-text available via subscription   (Followers: 16, SJR: 1.718, h-index: 58)
Advances in Physical Organic Chemistry     Full-text available via subscription   (Followers: 8, SJR: 0.384, h-index: 26)
Advances in Phytomedicine     Full-text available via subscription  
Advances in Planar Lipid Bilayers and Liposomes     Full-text available via subscription   (Followers: 3, SJR: 0.248, h-index: 11)
Advances in Plant Biochemistry and Molecular Biology     Full-text available via subscription   (Followers: 7)
Advances in Plant Pathology     Full-text available via subscription   (Followers: 5)
Advances in Porous Media     Full-text available via subscription   (Followers: 5)
Advances in Protein Chemistry     Full-text available via subscription   (Followers: 18)
Advances in Protein Chemistry and Structural Biology     Full-text available via subscription   (Followers: 20, SJR: 1.5, h-index: 62)
Advances in Quantum Chemistry     Full-text available via subscription   (Followers: 6, SJR: 0.478, h-index: 32)
Advances in Radiation Oncology     Open Access  
Advances in Small Animal Medicine and Surgery     Hybrid Journal   (Followers: 3, SJR: 0.1, h-index: 2)
Advances in Space Biology and Medicine     Full-text available via subscription   (Followers: 5)
Advances in Space Research     Full-text available via subscription   (Followers: 373, SJR: 0.606, h-index: 65)
Advances in Structural Biology     Full-text available via subscription   (Followers: 8)
Advances in Surgery     Full-text available via subscription   (Followers: 9, SJR: 0.823, h-index: 27)
Advances in the Study of Behavior     Full-text available via subscription   (Followers: 31, SJR: 1.321, h-index: 56)
Advances in Veterinary Medicine     Full-text available via subscription   (Followers: 16)
Advances in Veterinary Science and Comparative Medicine     Full-text available via subscription   (Followers: 13)
Advances in Virus Research     Full-text available via subscription   (Followers: 6, SJR: 1.878, h-index: 68)
Advances in Water Resources     Hybrid Journal   (Followers: 45, SJR: 2.408, h-index: 94)
Aeolian Research     Hybrid Journal   (Followers: 5, SJR: 0.973, h-index: 22)
Aerospace Science and Technology     Hybrid Journal   (Followers: 340, SJR: 0.816, h-index: 49)
AEU - Intl. J. of Electronics and Communications     Hybrid Journal   (Followers: 8, SJR: 0.318, h-index: 36)
African J. of Emergency Medicine     Open Access   (Followers: 6, SJR: 0.344, h-index: 6)
Ageing Research Reviews     Hybrid Journal   (Followers: 9, SJR: 3.289, h-index: 78)
Aggression and Violent Behavior     Hybrid Journal   (Followers: 437, SJR: 1.385, h-index: 72)
Agri Gene     Hybrid Journal  
Agricultural and Forest Meteorology     Hybrid Journal   (Followers: 15, SJR: 2.18, h-index: 116)
Agricultural Systems     Hybrid Journal   (Followers: 31, SJR: 1.275, h-index: 74)
Agricultural Water Management     Hybrid Journal   (Followers: 42, SJR: 1.546, h-index: 79)
Agriculture and Agricultural Science Procedia     Open Access  
Agriculture and Natural Resources     Open Access   (Followers: 3)
Agriculture, Ecosystems & Environment     Hybrid Journal   (Followers: 56, SJR: 1.879, h-index: 120)
Ain Shams Engineering J.     Open Access   (Followers: 5, SJR: 0.434, h-index: 14)
Air Medical J.     Hybrid Journal   (Followers: 5, SJR: 0.234, h-index: 18)
AKCE Intl. J. of Graphs and Combinatorics     Open Access   (SJR: 0.285, h-index: 3)
Alcohol     Hybrid Journal   (Followers: 11, SJR: 0.922, h-index: 66)
Alcoholism and Drug Addiction     Open Access   (Followers: 8)
Alergologia Polska : Polish J. of Allergology     Full-text available via subscription   (Followers: 1)
Alexandria Engineering J.     Open Access   (Followers: 1, SJR: 0.436, h-index: 12)
Alexandria J. of Medicine     Open Access   (Followers: 1)
Algal Research     Partially Free   (Followers: 9, SJR: 2.05, h-index: 20)
Alkaloids: Chemical and Biological Perspectives     Full-text available via subscription   (Followers: 3)
Allergologia et Immunopathologia     Full-text available via subscription   (Followers: 1, SJR: 0.46, h-index: 29)
Allergology Intl.     Open Access   (Followers: 4, SJR: 0.776, h-index: 35)
Alpha Omegan     Full-text available via subscription   (SJR: 0.121, h-index: 9)
ALTER - European J. of Disability Research / Revue Européenne de Recherche sur le Handicap     Full-text available via subscription   (Followers: 9, SJR: 0.158, h-index: 9)
Alzheimer's & Dementia     Hybrid Journal   (Followers: 49, SJR: 4.289, h-index: 64)
Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring     Open Access   (Followers: 4)
Alzheimer's & Dementia: Translational Research & Clinical Interventions     Open Access   (Followers: 4)
Ambulatory Pediatrics     Hybrid Journal   (Followers: 5)
American Heart J.     Hybrid Journal   (Followers: 48, SJR: 3.157, h-index: 153)
American J. of Cardiology     Hybrid Journal   (Followers: 48, SJR: 2.063, h-index: 186)
American J. of Emergency Medicine     Hybrid Journal   (Followers: 42, SJR: 0.574, h-index: 65)
American J. of Geriatric Pharmacotherapy     Full-text available via subscription   (Followers: 9, SJR: 1.091, h-index: 45)
American J. of Geriatric Psychiatry     Hybrid Journal   (Followers: 14, SJR: 1.653, h-index: 93)
American J. of Human Genetics     Hybrid Journal   (Followers: 32, SJR: 8.769, h-index: 256)
American J. of Infection Control     Hybrid Journal   (Followers: 26, SJR: 1.259, h-index: 81)
American J. of Kidney Diseases     Hybrid Journal   (Followers: 31, SJR: 2.313, h-index: 172)
American J. of Medicine     Hybrid Journal   (Followers: 45, SJR: 2.023, h-index: 189)
American J. of Medicine Supplements     Full-text available via subscription   (Followers: 3)
American J. of Obstetrics and Gynecology     Hybrid Journal   (Followers: 210, SJR: 2.255, h-index: 171)
American J. of Ophthalmology     Hybrid Journal   (Followers: 61, SJR: 2.803, h-index: 148)
American J. of Ophthalmology Case Reports     Open Access   (Followers: 6)
American J. of Orthodontics and Dentofacial Orthopedics     Full-text available via subscription   (Followers: 6, SJR: 1.249, h-index: 88)
American J. of Otolaryngology     Hybrid Journal   (Followers: 24, SJR: 0.59, h-index: 45)
American J. of Pathology     Hybrid Journal   (Followers: 27, SJR: 2.653, h-index: 228)
American J. of Preventive Medicine     Hybrid Journal   (Followers: 26, SJR: 2.764, h-index: 154)
American J. of Surgery     Hybrid Journal   (Followers: 36, SJR: 1.286, h-index: 125)
American J. of the Medical Sciences     Hybrid Journal   (Followers: 12, SJR: 0.653, h-index: 70)
Ampersand : An Intl. J. of General and Applied Linguistics     Open Access   (Followers: 6)
Anaerobe     Hybrid Journal   (Followers: 4, SJR: 1.066, h-index: 51)
Anaesthesia & Intensive Care Medicine     Full-text available via subscription   (Followers: 60, SJR: 0.124, h-index: 9)
Anaesthesia Critical Care & Pain Medicine     Full-text available via subscription   (Followers: 14)
Anales de Cirugia Vascular     Full-text available via subscription  
Anales de Pediatría     Full-text available via subscription   (Followers: 2, SJR: 0.209, h-index: 27)
Anales de Pediatría (English Edition)     Full-text available via subscription  
Anales de Pediatría Continuada     Full-text available via subscription   (SJR: 0.104, h-index: 3)
Analytic Methods in Accident Research     Hybrid Journal   (Followers: 4, SJR: 2.577, h-index: 7)
Analytica Chimica Acta     Hybrid Journal   (Followers: 36, SJR: 1.548, h-index: 152)
Analytical Biochemistry     Hybrid Journal   (Followers: 174, SJR: 0.725, h-index: 154)
Analytical Chemistry Research     Open Access   (Followers: 8, SJR: 0.18, h-index: 2)
Analytical Spectroscopy Library     Full-text available via subscription   (Followers: 12)
Anesthésie & Réanimation     Full-text available via subscription   (Followers: 1)
Anesthesiology Clinics     Full-text available via subscription   (Followers: 22, SJR: 0.421, h-index: 40)
Angiología     Full-text available via subscription   (SJR: 0.124, h-index: 9)
Angiologia e Cirurgia Vascular     Open Access  
Animal Behaviour     Hybrid Journal   (Followers: 178, SJR: 1.907, h-index: 126)
Animal Feed Science and Technology     Hybrid Journal   (Followers: 5, SJR: 1.151, h-index: 83)

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Journal Cover Analytical Biochemistry
  [SJR: 0.725]   [H-I: 154]   [174 followers]  Follow
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0003-2697 - ISSN (Online) 1096-0309
   Published by Elsevier Homepage  [3123 journals]
  • Drone inflight mixing of biochemical samples
    • Authors: Mayur Katariya; Dwayne Chung Kim Chung; Tristan Minife; Harshit Gupta; Alifa Afiah Ahmad Zahidi; Oi Wah Liew; Tuck Wah Ng
      Pages: 1 - 3
      Abstract: Publication date: 15 March 2018
      Source:Analytical Biochemistry, Volume 545
      Author(s): Mayur Katariya, Dwayne Chung Kim Chung, Tristan Minife, Harshit Gupta, Alifa Afiah Ahmad Zahidi, Oi Wah Liew, Tuck Wah Ng
      Autonomous systems for sample transport to the laboratory for analysis can be improved in terms of timeliness, cost and error mitigation in the pre-analytical testing phase. Drones have been reported for outdoor sample transport but incorporating devices on them to attain homogenous mixing of reagents during flight to enhance sample processing timeliness is limited by payload issues. It is shown here that flipping maneuvers conducted with quadcopters are able to facilitate complete and gentle mixing. This capability incorporated during automated sample transport serves to address an important factor contributing to pre-analytical variability which ultimately impacts on test result reliability.
      Graphical abstract image

      PubDate: 2018-01-26T05:14:01Z
      DOI: 10.1016/j.ab.2018.01.004
      Issue No: Vol. 545 (2018)
  • Multiplexed isothermal nucleic acid amplification
    • Authors: Olena Mayboroda; Ioanis Katakis; Ciara K. O'Sullivan
      Pages: 20 - 30
      Abstract: Publication date: 15 March 2018
      Source:Analytical Biochemistry, Volume 545
      Author(s): Olena Mayboroda, Ioanis Katakis, Ciara K. O'Sullivan
      Multiplexed isothermal amplification and detection of nucleic acid sequences and biomarkers is of increasing importance in diverse areas including advanced diagnostics, food quality control and environmental monitoring. Whilst there are several very elegant isothermal amplification approaches, multiplexed amplification remains a challenge, requiring careful experimental design and optimisation, from judicious primer design in order to avoid the formation of primer dimers and non-specific amplification, applied temperature as well as the ratio and concentration of primers. In this review, we describe the various approaches that have been reported to date for multiplexed isothermal amplification, for both “one-pot” multiplexing as well as parallelised multiplexing using loop-mediated isothermal amplification, strand-displacement amplification, helicase-dependent amplification, rolling circle amplification, nucleic acid sequence-based amplification, with a particular focus on recombinase polymerase amplification.

      PubDate: 2018-01-26T05:14:01Z
      DOI: 10.1016/j.ab.2018.01.005
      Issue No: Vol. 545 (2018)
  • Rapid detection of foodborne pathogen Listeria monocytogenes by strand
           exchange amplification
    • Authors: Meiling Zhang; Xiudan Wang; Lingzhi Han; Shuyan Niu; Chao Shi; Cuiping Ma
      Pages: 38 - 42
      Abstract: Publication date: 15 March 2018
      Source:Analytical Biochemistry, Volume 545
      Author(s): Meiling Zhang, Xiudan Wang, Lingzhi Han, Shuyan Niu, Chao Shi, Cuiping Ma
      A strand exchange amplification (SEA) method to detect foodborne pathogen Listeria monocytogenes was developed. SEA is a novel nucleic acid amplification method that only requires one pair of primers. The specie-specific primers were designed by targeting the 16S rRNA gene and the amplification reaction was performed as short as 60 min at 61 °C. Notably, SEA method could not only detect genomic DNA but also detect RNA by one step without requiring extra reverse transcription. The result could be visualized by naked eyes so that water bath pot would be the only equipment needed. Moreover, culture fluids and bacteria colony could be successfully detected without any pretreatment and the method displayed good specificity and strong anti-jamming capacity. These features greatly simplified the operating procedure and made SEA method be potential for developing point-of-care testing (POCT) devices to detect viable L. monocytogenes.

      PubDate: 2018-01-26T05:14:01Z
      DOI: 10.1016/j.ab.2018.01.013
      Issue No: Vol. 545 (2018)
  • Paper-based detection of HIV-1 drug resistance using isothermal
           amplification and an oligonucleotide ligation assay
    • Authors: Mary E. Natoli; Brittany A. Rohrman; Carolina De Santiago; Gert U. van Zyl; Rebecca R. Richards-Kortum
      Pages: 64 - 71
      Abstract: Publication date: 1 March 2018
      Source:Analytical Biochemistry, Volume 544
      Author(s): Mary E. Natoli, Brittany A. Rohrman, Carolina De Santiago, Gert U. van Zyl, Rebecca R. Richards-Kortum
      Regular HIV-1 viral load monitoring is the standard of care to assess antiretroviral therapy effectiveness in resource-rich settings. Persistently elevated viral loads indicate virologic failure (VF), which warrants HIV drug resistance testing (HIVDRT) to allow individualized regimen switches. However, in settings lacking access to HIVDRT, clinical decisions are often made based on symptoms, leading to unnecessary therapy switches and increased costs of care. This work presents a proof-of-concept assay to detect M184V, the most common drug resistance mutation after first-line antiretroviral therapy failure, in a paper format. The first step isothermally amplifies a section of HIV-1 reverse transcriptase containing M184V using a recombinase polymerase amplification (RPA) assay. Then, an oligonucleotide ligation assay (OLA) is used to selectively label the mutant and wild type amplified sequences. Finally, a lateral flow enzyme-linked immunosorbent assay (ELISA) differentiates between OLA-labeled products with or without M184V. Our method shows 100% specificity and 100% sensitivity when tested with samples that contained 200 copies of mutant DNA and 800 copies of wild type DNA prior to amplification. When integrated with sample preparation, this method may detect HIV-1 drug resistance at a low cost and at a rural hospital laboratory.
      Graphical abstract image

      PubDate: 2018-01-04T09:57:39Z
      DOI: 10.1016/j.ab.2017.12.008
      Issue No: Vol. 544 (2018)
  • Application of elastin-based nanoparticles displaying antibody binding
           domains for a homogeneous immunoassay
    • Authors: Tsutomu Sugihara; Masayasu Mie; Eiry Kobatake
      Pages: 72 - 79
      Abstract: Publication date: 1 March 2018
      Source:Analytical Biochemistry, Volume 544
      Author(s): Tsutomu Sugihara, Masayasu Mie, Eiry Kobatake
      Nanoparticles are small size-controlled particles from 1 to 100 nm diameters and characterized by their structure, base material and functional units displayed on their surfaces. In this study, protein-based nanoparticles composed of a hydrophobic elastin-like peptide unit, a hydrophilic aspartic acid-rich peptide unit and displaying antibody binding domains on their surfaces, were designed and genetically synthesized. The constituent fusion proteins, termed ELP-D-C, were found to exist in monomeric form (ELP-D-C/monomer) at low temperature. Above the phase transition temperature, however, ELP-D-C was found to rapidly self-assemble to form spherical micelles (ELP-D-C/micelle) with a hydrophobic core and diameters of ∼40 nm. Furthermore, ELP-D-C/micelle were shown to display antibody binding domains on their surfaces, which allowed for immobilization of antibodies and subsequent formation of large, visually detectable complexes in the presence of target molecule (antigen), whose sizes increased in proportion to the target molecule concentration. The observed target molecule concentration-dependent complex formation suggests that ELP-D-C/micelle may be useful as base particles in applications such as homogeneous turbidity immunoassays.

      PubDate: 2018-01-04T09:57:39Z
      DOI: 10.1016/j.ab.2017.12.023
      Issue No: Vol. 544 (2018)
  • A continuous assay for l-talarate/galactarate dehydratase using circular
    • Authors: Nicole M. Easton; Sarah A.E. Aboushawareb; Stephen L. Bearne
      Pages: 80 - 86
      Abstract: Publication date: 1 March 2018
      Source:Analytical Biochemistry, Volume 544
      Author(s): Nicole M. Easton, Sarah A.E. Aboushawareb, Stephen L. Bearne
      l-Talarate/galactarate dehydratase (TGD) is a member of the enolase superfamily of enzymes and catalyzes the dehydration of either meso-galactarate or l-talarate to form 5-keto-4-deoxy-d-glucarate (5-KDG). To facilitate study of this enzyme and other galactarate dehydratases, a continuous circular dichroism-based assay has been developed. Using recombinant enzyme from Salmonella typhimurium (StTGD), the rates of StTGD-catalyzed conversion of m-galactarate to 5-KDG were determined by following the change in ellipticity at 323 nm. The apparent molar ellipticity ([θ]323) for the 5-KDG formed was determined to be 202 ± 2 deg cm2 dmol−1, which was used to convert observed rates (Δθ/Δt) into concentration-dependent rates (Δc/Δt). The kinetic parameters K m, k cat, and k cat/K m were 0.38 ± 0.05 mM, 4.8 ± 0.1 s−1, and 1.3 (±0.2) × 104 M−1s−1, respectively. These values are in excellent agreement with those published previously [Yew, W.S. et al. (2007) Biochemistry 46, 9564–9577] using a coupled assay system. To demonstrate the utility of the assay, the inhibition constant (K i = 10.7 ± 0.4 mM) was determined for the competitive inhibitor tartronate. The continuous CD-based assay offers a practical and efficient alternative method to the coupled assay that requires access to 5-KDG aldolase, and to the labor-intensive, fixed-time assays.
      Graphical abstract image

      PubDate: 2018-01-04T09:57:39Z
      DOI: 10.1016/j.ab.2017.12.015
      Issue No: Vol. 544 (2018)
  • A microfluidic enrichment platform with a recombinase polymerase
           amplification sensor for pathogen diagnosis
    • Authors: Thuy Nguyen Thi Dao; Eun Yeong Lee; Bonhan Koo; Choong Eun Jin; Tae Yoon Lee; Yong Shin
      Pages: 87 - 92
      Abstract: Publication date: 1 March 2018
      Source:Analytical Biochemistry, Volume 544
      Author(s): Thuy Nguyen Thi Dao, Eun Yeong Lee, Bonhan Koo, Choong Eun Jin, Tae Yoon Lee, Yong Shin
      Rapid and sensitive detection of low amounts of pathogen in large samples is needed for early diagnosis and treatment of patients and surveillance of pathogen. In this study, we report a microfluidic platform for detection of low pathogen levels in a large sample volume that couples an Magainin 1 based microfluidic platform for pathogen enrichment and a recombinase polymerase amplification (RPA) sensor for simultaneous pathogenic DNA amplification and detection in a label-free and real-time manner. Magainin 1 is used as a pathogen enrichment agent with a herringbone microfluidic chip. Using this enrichment platform, the detection limit was found to be 20 times more sensitive in 10 ml urine with Salmonella and 10 times more sensitive in 10 ml urine with Brucella than that of real-time PCR without the enrichment process. Furthermore, the combination system of the enrichment platform and an RPA sensor that based on an isothermal DNA amplification method with rapidity and sensitivity for detection can detect a pathogen at down to 50 CFU in 10 ml urine for Salmonella and 102 CFU in 10 ml urine for Brucella within 60 min. This system will be useful as it has the potential for better diagnosis of pathogens by increasing the capture efficiency of the pathogen in large samples, subsequently enhancing the detection limit of pathogenic DNA.

      PubDate: 2018-01-10T12:17:30Z
      DOI: 10.1016/j.ab.2017.12.030
      Issue No: Vol. 544 (2018)
  • False positives in using the zymogram assay for identification of
           peptidoglycan hydrolases
    • Authors: Cristian A. Escobar; Timothy A. Cross
      Pages: 162 - 166
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Cristian A. Escobar, Timothy A. Cross
      Zymogram assays have been used extensively to identify novel peptidoglycan hydrolases. In this study it is reported that the zymogram is susceptible to false positive results when highly positively charged proteins are assayed. As an example, we report on the case of the ChiZ membrane protein from the Mycobacterium tuberculosis divisome, which previously was described as a peptidoglycan hydrolase. Even though the full length ChiZ protein was able to produce positive assay results, other direct methods for measuring peptidoglycan hydrolysis do not provide convincing evidence that ChiZ has peptidoglycan hydrolysis activity. We show that the false positive result is produced by the highly positively charged N-terminal region of ChiZ. Thus, we developed a zymogram control that can be used to identify false positives results. This control assay lacks the refolding step in the normal zymogram assay. For lysozyme the control assay shows no activity, while the N-terminal region of ChiZ shows a false positive result. Given the limitations of the zymogram assay to reliably identify peptidoglycan hydrolases, we recommend using the zymogram control assay together with other methods to evaluate possible peptidoglycan hydrolysis activity.

      PubDate: 2018-01-04T09:57:39Z
      DOI: 10.1016/j.ab.2017.12.016
      Issue No: Vol. 543 (2018)
  • Microwave-assisted enzymatic hydrolysis of DNA for mass spectrometric
           analysis: A new strategy for accelerated hydrolysis
    • Abstract: Publication date: 1 April 2018
      Source:Analytical Biochemistry, Volume 546
      Author(s): Sujatha Chilakala, Lan Li, Ye Feng, Yan Xu
      Study of DNA base composition, DNA adducts and modification in its primary structure is a subject of interest in different fields of scientific research. Various methods like immunochemistry, capillary electrophoretic separation, chromatographic separation coupled with mass spectrometric (LC-MS/MS) detection have been developed for DNA analysis. During the past decade LC-MS/MS has emerged as a more sensitive and selective technique and now frequently used for the analysis of DNA. The workflow for the DNA analysis include DNA extraction, hydrolysis of DNA into mononucleosides and analysis. Though high-throughput methods are available for the analysis DNA hydrolysis oftentimes is a rate limiting step. Conventional enzymatic hydrolysis of DNA using a multienzyme mixture will take a minimum of 6–17 h to complete the hydrolysis. In this work, we developed an accelerated enzymatic hydrolysis of DNA using microwave-technology for the first time. 1.00 μg of calf thymus DNA was used to demonstrate the microwave assisted enzymatic hydrolysis of DNA. The resultant mononucleosides were separated on Atlantis T3 (50 × 2.1 mm i.d.; 3 μ particle size) C18 column and analyzed on ABSCIEX QTRAP 5500 LC/MS system. The sample through put and recovery of microwave-assisted digestion were compared with the conventional enzymatic hydrolysis. Efficient digestion of DNA with a performance similar to that obtained by the conventional overnight digestion procedure was attained in just 30 min with a hydrolysis yield of ≥90%. Furthermore, our method was found to be much more accurate and easier to perform. Thus, this new application of microwave technology to DNA enzymatic digestion will facilitate the application of DNA analysis in biological and clinical research.

      PubDate: 2018-02-15T00:02:22Z
  • Kinetic studies of serine protease inhibitors in simple and rapid
           ‘active barrier’ model systems - Diffusion through an inhibitor
    • Abstract: Publication date: 1 April 2018
      Source:Analytical Biochemistry, Volume 546
      Author(s): Erika Billinger, Gunnar Johansson
      A model based on gelatin for protease activity studies was designed. The model is also extended to study the efficiency of inhibitors in a separate protective layer covering the layer containing the target substrate. A good correlation between protease concentration and the size of erosion wells formed in a plain gelatin layer was observed. Similarly, increased concentration of inhibitors gave a systematic decrease in well area. Kinetic analyses of the two-layer model in a spectrophotometric plate reader with a fixed concentration of substrate in the bottom layer displayed a strict dependence of both inhibitor concentration and thickness of the top “protective” layer. An apparent, but weaker inhibition effect was also observed without inhibitors due to diffusional and erosion delay of enzyme transport to the substrate-containing layer.

      PubDate: 2018-02-15T00:02:22Z
  • Characterization of fatty acid amide hydrolase activity by a
           fluorescence-based assay
    • Abstract: Publication date: 1 April 2018
      Source:Analytical Biochemistry, Volume 546
      Author(s): Florian M. Dato, Andreas Maaßen, Bernd Goldfuß, Markus Pietsch
      Fatty acid amide hydrolase (FAAH) is involved in many human diseases, particularly cancer, pain and inflammation as well as neurological, metabolic and cardiovascular disorders. Therefore, FAAH is an attractive target for the development of low-molecular-weight inhibitors as therapeutics, which requires robust assays that can be used for high-throughput screening (HTS) of compound libraries. Here, we report the development of a fluorometric assay based on FAAH's ability to effectively hydrolyze medium-chain fatty acid amides, introducing N-decanoyl-substituted 5-amino-2-methoxypyridine (D-MAP) as new amide substrate. D-MAP is cleaved by FAAH with an 8-fold larger specificity constant than the previously reported octanoyl-analog Oc-MAP (Vmax/K m of 1.09 and 0.134 mL min−1 mg−1, respectively), with both MAP derivatives possessing superior substrate properties and much increased aqueous solubility compared to the respective p-nitroaniline compounds D-pNA and Oc-pNA. The new assay with D-MAP as substrate is highly sensitive using a lower enzyme concentration (1 μg mL−1) than literature-reported fluorimetric FAAH assays. In addition, D-MAP was validated in comparison to the substrate Oc-MAP for the characterization of FAAH inhibitors by means of the reference compounds URB597 and TC-F2 and was shown to be highly suitable for HTS in both kinetic and endpoint assays (Z′ factors of 0.81 and 0.78, respectively).

      PubDate: 2018-02-15T00:02:22Z
  • Heterogeneous asymmetric recombinase polymerase amplification (haRPA) for
           rapid hygiene control of large-volume water samples
    • Abstract: Publication date: 1 April 2018
      Source:Analytical Biochemistry, Volume 546
      Author(s): Dennis Elsäßer, Johannes Ho, Reinhard Niessner, Andreas Tiehm, Michael Seidel
      Hygiene of drinking water is periodically controlled by cultivation and enumeration of indicator bacteria. Rapid and comprehensive measurements of emerging pathogens are of increasing interest to improve drinking water safety. In this study, the feasibility to detect bacteriophage PhiX174 as a potential indicator for virus contamination in large volumes of water is demonstrated. Three consecutive concentration methods (continuous ultrafiltration, monolithic adsorption filtration, and centrifugal ultrafiltration) were combined to concentrate phages stepwise from 1250 L drinking water into 1 mL. Heterogeneous asymmetric recombinase polymerase amplification (haRPA) is applied as rapid detection method. Field measurements were conducted to test the developed system for hygiene online monitoring under realistic conditions. We could show that this system allows the detection of artificial contaminations of bacteriophage PhiX174 in drinking water pipelines.
      Graphical abstract image

      PubDate: 2018-02-15T00:02:22Z
  • Development of a lateral flow recombinase polymerase assay for the
           diagnosis of Schistosoma mansoni infections
    • Abstract: Publication date: 1 April 2018
      Source:Analytical Biochemistry, Volume 546
      Author(s): Kate Poulton, Bonnie Webster
      Infection with Schistosoma mansoni causes intestinal schistosomiasis, a major health problem across Africa. The accurate diagnosis of intestinal schistosomiasis is vital to inform surveillance/control programs. Diagnosis mainly relies on microscopic detection of eggs in faecal samples but many factors affect sensitivity. Molecular diagnostics are sensitive and specific but application is limited as necessary infrastructure, financial resources and skilled personnel are often lacking in endemic settings. Recombinase Polymerase Amplification (RPA) is an isothermal DNA amplification/detection technology that is practical in nearly any setting. Here we developed a RPA lateral flow (LF) assay targeting the 28S rDNA region of S. mansoni. The 28S LF-RPA assay's lower limit of detection was 10pg DNA with the lower test parameters permitting sufficient amplification being 6 min and 25°C. Optimal assay parameters were 40–45°C and 10 min with an analytical sensitivity of 102 copies of DNA. Additionally the PCRD3 lateral flow detection cassettes proved more robust and sensitive compared to the Milenia HybriDetect strips. This 28S LF-RPA assay produces quick reproducible results that are easy to interpret, require little infrastructure and is a promising PON test for the field molecular diagnosis of intestinal schistosomiasis.

      PubDate: 2018-02-15T00:02:22Z
  • Recombinase polymerase amplification applied to plant virus detection and
           potential implications
    • Abstract: Publication date: 1 April 2018
      Source:Analytical Biochemistry, Volume 546
      Author(s): Binoy Babu, Francisco M. Ochoa-Corona, Mathews L. Paret
      Several isothermal techniques for the detection of plant pathogens have been developed with the advent of molecular techniques. Among them, Recombinase Polymerase Amplification (RPA) is becoming an important technique for the rapid, sensitive and cost-effective detection of plant viruses. The RPA technology has the advantage to be implemented in field-based scenarios because the method requires a minimal sample preparation, and is performed at constant low temperature (37–42 °C). The RPA technique is rapidly becoming a promising tool for use in rapid detection and further diagnostics in plant clinics and monitoring quarantine services. This paper presents a review of studies conducted using RPA for detection/diagnosis of plant viruses with either DNA genomes (Banana bunchy top virus, Bean golden yellow mosaic virus, Tomato mottle virus, Tomato yellow leaf curl virus) or RNA genomes (Little Cherry virus 2, Plum pox virus and Rose rosette virus).

      PubDate: 2018-02-15T00:02:22Z
  • Zeolitic imidazolate framework-based biosensor for detection of HIV-1 DNA
    • Abstract: Publication date: 1 April 2018
      Source:Analytical Biochemistry, Volume 546
      Author(s): Yong Pan, Shenshan Zhan, Fan Xia
      ZIF-8(zinc-methylimidazolate framework-8), one of the zeolitic imidazolate frameworks (ZIFs), as quenching platforms for detection of HIV-1 DNA, which was identified to be effective for highly sensitive detection of HIV-1 DNA. The enhanced fluorescence signal has a relationship with the ssDNA concentration, the detection limit is as low as 1.2 nmol L−1 with good selectivity. This study is an important step toward rational design of materials to achieve specific interactions between biomolecules and synthetic particle surfaces.

      PubDate: 2018-02-05T02:18:34Z
  • A simple and effective method for detecting precipitated proteins in
           MALDI−TOF MS
    • Abstract: Publication date: 1 April 2018
      Source:Analytical Biochemistry, Volume 546
      Author(s): Hiroyuki Oshikane, Masahiko Watabe, Toshio Nakaki
      MALDI−TOF MS has developed rapidly into an essential analytical tool for the life sciences. Cinnamic acid derivatives are generally employed in routine molecular weight determinations of intact proteins using MALDI−TOF MS. However, a protein of interest may precipitate when mixed with matrix solution, perhaps preventing MS detection. We herein provide a simple approach to enable the MS detection of such precipitated protein species by means of a “direct deposition method” -- loading the precipitant directly onto the sample plate. It is thus expected to improve routine MS analysis of intact proteins.

      PubDate: 2018-02-05T02:18:34Z
  • Reactivity-driven cleanup of 2-Aminobenzamide derivatized oligosaccharides
    • Abstract: Publication date: 1 April 2018
      Source:Analytical Biochemistry, Volume 546
      Author(s): An-Hsiang Adam Chu, Andrew E. Saati, John J. Scarcelli, Richard J. Cornell, Thomas J. Porter
      N-glycan profiling is commonly accomplished by the derivatization of the enzymatically released oligosaccharides with a fluorophore, thereby facilitating their analysis by hydrophilic-interaction liquid chromatography (HILIC). These fluorescent dyes are often present in large excess during derivatization reactions, and their removal is typically required to minimize chromatographic interference. Herein, we report a reactivity-driven 2-phase extraction protocol with the aldehyde reagent octanal, which demonstrated efficient 2-aminobenzamide cleanup as well as high derivatized N-glycan recovery. This cleanup method can be performed within minutes, and therefore provides an alternative sample preparation route for N-glycan profiling with improved time efficiency and operational simplicity.
      Graphical abstract image

      PubDate: 2018-02-05T02:18:34Z
  • Duplex recombinase polymerase amplification assays incorporating
           competitive internal controls for bacterial meningitis detection
    • Abstract: Publication date: 1 April 2018
      Source:Analytical Biochemistry, Volume 546
      Author(s): Owen Higgins, Eoin Clancy, Matthew S. Forrest, Olaf Piepenburg, Martin Cormican, Teck Wee Boo, Nicola O'Sullivan, Claire McGuinness, Deirdre Cafferty, Robert Cunney, Terry J. Smith
      Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technology that provides rapid and robust infectious disease pathogen detection, ideal for point-of-care (POC) diagnostics in disease-prevalent low-resource countries. We have developed and evaluated three duplex RPA assays incorporating competitive internal controls for the detection of leading bacterial meningitis pathogens. Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae singleplex RPA assays were initially developed and evaluated, demonstrating 100% specificity with limits of detection of 4.1, 8.5 and 3.9 genome copies per reaction, respectively. Each assay was further developed into internally controlled duplex RPA assays via the incorporation of internal amplification control templates. Clinical performance of each internally controlled duplex RPA assay was evaluated by testing 64 archived PCR-positive clinical samples. Compared to real-time PCR, all duplex RPA assays demonstrated 100% diagnostic specificity, with diagnostic sensitivities of 100%, 86.3% and 100% for the S. pneumoniae, N. meningitidis and H. influenzae assays, respectively. This study details the first report of internally controlled duplex RPA assays for the detection of bacterial meningitis pathogens: S. pneumoniae, N. meningitidis and H. influenzae. We have successfully demonstrated the clinical diagnostic utility of each duplex RPA assay, introducing effective diagnostic technology for POC bacterial meningitis identification in disease-prevalent developing countries.

      PubDate: 2018-02-05T02:18:34Z
  • Rapid detection of potyviruses from crude plant extracts
    • Abstract: Publication date: 1 April 2018
      Source:Analytical Biochemistry, Volume 546
      Author(s): Gonçalo Silva, Joshua Oyekanmi, Chukwuemeka K. Nkere, Moritz Bömer, P. Lava Kumar, Susan E. Seal
      Potyviruses (genus Potyvirus; family Potyviridae) are widely distributed and represent one of the most economically important genera of plant viruses. Therefore, their accurate detection is a key factor in developing efficient control strategies. However, this can sometimes be problematic particularly in plant species containing high amounts of polysaccharides and polyphenols such as yam (Dioscorea spp.). Here, we report the development of a reliable, rapid and cost-effective detection method for the two most important potyviruses infecting yam based on reverse transcription-recombinase polymerase amplification (RT-RPA). The developed method, named ‘Direct RT-RPA’, detects each target virus directly from plant leaf extracts prepared with a simple and inexpensive extraction method avoiding laborious extraction of high-quality RNA. Direct RT-RPA enables the detection of virus-positive samples in under 30 min at a single low operation temperature (37 °C) without the need for any expensive instrumentation. The Direct RT-RPA tests constitute robust, accurate, sensitive and quick methods for detection of potyviruses from recalcitrant plant species. The minimal sample preparation requirements and the possibility of storing RPA reagents without cold chain storage, allow Direct RT-RPA to be adopted in minimally equipped laboratories and with potential use in plant clinic laboratories and seed certification facilities worldwide.

      PubDate: 2018-02-05T02:18:34Z
  • Carboxylic group riched graphene oxide based disposable electrochemical
           immunosensor for cancer biomarker detection
    • Abstract: Publication date: 15 March 2018
      Source:Analytical Biochemistry, Volume 545
      Author(s): Sajid Rauf, Geetesh K. Mishra, Jahanzaib Azhar, Rupesh K. Mishra, K. Yugender Goud, Muhammad Azhar Hayat Nawaz, Jean Louis Marty, Akhtar Hayat
      In this work, we have developed for the first time a carboxylic group riched graphene oxide based disposable electrochemical immunosensor for cancer biomarker detection using methylene blue (MB). The developed immunosensor is highly sensitive for detection of biomarker Mucin1 (MUC1) in human serum samples. Development of this disposable electrochemical immunosensor was premeditated by applying specific monoclonal antibodies against MUC1. In this method, we explored highly conductive surface of carboxylic group (-COOH-) rich graphene oxide (GO) on screen-printed carbon electrodes (SPCE). This modified GO-COOH-SPCE was employed for the detection of MUC1 protein based on the reaction with methylene blue (MB) redox probe using differential pulse voltammetry (DPV) technique. Developed immunosensor exhibited good detection range for MUC1 with excellent linearity (0.1 U/mL- 2 U/mL), with a limit of detection of 0.04 U/mL. Upon potential application of developed biosensor, good recoveries were recorded in the range of 96–96.67% with % R.S.D 4.2. Analytical performance of the developed immunosensor assures the applicability in clinical diagnostic applications.
      Graphical abstract image

      PubDate: 2018-02-05T02:18:34Z
  • Novel volumetric adsorptive microsampling technique for determination of
           perfluorinated compounds in blood
    • Abstract: Publication date: 15 March 2018
      Source:Analytical Biochemistry, Volume 545
      Author(s): Jani Koponen, James Rudge, Stuart Kushon, Hannu Kiviranta
      Microsampling is an attractive option for significantly reducing the volume of blood taken for chemical analysis allowing for blood samples taken as a ‘finger-prick’ with a lancet. A novel, volumetric adsorptive microsampling (VAMS™) device, which reproducibly collects a small volume of 10 μL whole blood in a hematocrit-independent manner, is evaluated in a human biomonitoring setting, and has been utilized for analysis of several perfluoroalkyl acids (PFAA). The results show that the VAMS technique is applicable for PFAA analysis, method has good linearity, repeatability, accuracy and is sufficiently sensitive for samples from general populations. The stability of PFAAs with VAMS devices is shown to be acceptable, which supports the sampling and transportation strategy of several study designs. Furthermore, as well as allowing for a quick and efficient extraction and analysis flow path, the VAMS microsampler is an easy to use device in a real-world sample collection scenario.

      PubDate: 2018-02-05T02:18:34Z
  • Moving toward rapid and low-cost point-of-care molecular diagnostics with
           a repurposed 3D printer and RPA
    • Abstract: Publication date: 15 March 2018
      Source:Analytical Biochemistry, Volume 545
      Author(s): Kamfai Chan, Pui-Yan Wong, Chaitanya Parikh, Season Wong
      Traditionally, the majority of nucleic acid amplification-based molecular diagnostic tests are done in centralized settings. In recent years, point-of-care tests have been developed for use in low-resource settings away from central laboratories. While most experts agree that point-of-care molecular tests are greatly needed, their availability as cost-effective and easy-to-operate tests remains an unmet goal. In this article, we discuss our efforts to develop a recombinase polymerase amplification reaction-based test that will meet these criteria. First, we describe our efforts in repurposing a low-cost 3D printer as a platform that can carry out medium-throughput, rapid, and high-performing nucleic acid extraction. Next, we address how these purified templates can be rapidly amplified and analyzed using the 3D printer's heated bed or the deconstructed, low-cost thermal cycler we have developed. In both approaches, real-time isothermal amplification and detection of template DNA or RNA can be accomplished using a low-cost portable detector or smartphone camera. Last, we demonstrate the capability of our technologies using foodborne pathogens and the Zika virus. Our low-cost approach does not employ complicated and high-cost components, making it suitable for resource-limited settings. When integrated and commercialized, it will offer simple sample-to-answer molecular diagnostics.

      PubDate: 2018-02-05T02:18:34Z
  • Automated real-time detection of drug-resistant Mycobacterium tuberculosis
           on a lab-on-a-disc by Recombinase Polymerase Amplification
    • Authors: I.L.G. Law; J.F.C. Loo; H.C. Kwok; H.Y. Yeung; C.C.H. Leung; M. Hui; S.Y. Wu; H.S. Chan; Y.W. Kwan; H.P. Ho; S.K. Kong
      Abstract: Publication date: Available online 2 January 2018
      Source:Analytical Biochemistry
      Author(s): I.L.G. Law, J.F.C. Loo, H.C. Kwok, H.Y. Yeung, C.C.H. Leung, M. Hui, S.Y. Wu, H.S. Chan, Y.W. Kwan, H.P. Ho, S.K. Kong
      With the emergence of multi- and extensive-drug (MDR/XDR) resistant Mycobacterium tuberculosis (M. tb), tuberculosis (TB) persists as one of the world's leading causes of death. Recently, isothermal DNA amplification methods received much attention due to their ease of translation onto portable point-of-care (POC) devices for TB diagnosis. In this study, we aimed to devise a simple yet robust detection method for M. tb. Amongst the numerous up-and-coming isothermal techniques, Recombinase Polymerase Amplification (RPA) was chosen for a real-time detection of TB with or without MDR. In our platform, real-time RPA (RT-RPA) was integrated on a lab-on-a-disc (LOAD) with on-board power to maintain temperature for DNA amplification. Sputa collected from healthy volunteers were spiked with respective target M. tb samples for testing. A limit of detection of 102 colony-forming unit per millilitre in 15 min was achieved, making early detection and differentiation of M. tb strains highly feasible in extreme POC settings. Our RT-RPA LOAD platform has also been successfully applied in the differentiation of MDR-TB from H37Ra, an attenuated TB strain. In summary, a quantitative RT-RPA on LOAD assay with a high level of sensitivity was developed as a foundation for further developments in medical bedside and POC diagnostics.

      PubDate: 2018-01-04T09:57:39Z
      DOI: 10.1016/j.ab.2017.12.031
  • Selective recognition of creatinine – Development of a colorimetric
    • Authors: Unni Sivasankaran; Theresa Chiramal Jos; Krishnapillai Girish Kumar
      Pages: 1 - 6
      Abstract: Publication date: 1 March 2018
      Source:Analytical Biochemistry, Volume 544
      Author(s): Unni Sivasankaran, Theresa Chiramal Jos, Krishnapillai Girish Kumar
      The present report describes a simple and cost effective protocol for colourimetric determination of creatinine (CR), L-cysteine stabilized copper nanoparticles (L-cys-CuNPs) exhibited selective and sensitive interaction with CR. Utilizing this interaction, a colourimetric sensor has been developed based on the reduction in LSPR intensity as monitored by a UV–visible spectrophotometer. The developed sensor exhibited a linear dynamic range of 5.33 × 10−6 to 3.33 × 10−7 M. Proposed sensor is simple and cost - effective compared to methods based on noble metal nanoparticles and the sensitivity to determine CR was as low as 4.54 × 10−10 M. The sensor was successfully applied for quantification of CR in artificial serum and urine samples. Sensor developed in this work has a high potential for rapid and on-site determination of CR in physiological and clinical samples.
      Graphical abstract image

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.017
      Issue No: Vol. 544 (2017)
  • Glassy carbon electrode modified with carbon black for sensitive estradiol
           determination by means of voltammetry and flow injection analysis with
           amperometric detection
    • Authors: Joanna Smajdor; Robert Piech; Martyna Ławrywianiec; Beata Paczosa-Bator
      Pages: 7 - 12
      Abstract: Publication date: 1 March 2018
      Source:Analytical Biochemistry, Volume 544
      Author(s): Joanna Smajdor, Robert Piech, Martyna Ławrywianiec, Beata Paczosa-Bator
      A voltammetric method for fast and sensitive estradiol determination using carbon black modified glassy carbon electrode (CBGC) is proposed. The use of carbon black as a modifying layer led to obtain low detection limit (9.2·10−8 mol L−1 for a preconcentration time of 60 s) and stability of registered signals (measured as RSD is 1.3%, n = 7, estradiol concentration 0.5·10−6 mol L−1). Cyclic voltammetry study revealed that in phosphate media estradiol suffers irreversible one-proton and one-electron oxidation process. Under the optimum conditions, estradiol calibration curve was linear in the concentration range from 0.15·10−6 to 3.5·10−6 mol L−1. The proposed method enable to determine estradiol content in different pharmaceutical formulation with good recovery. Amperometric measurements of estradiol were performed as well to indicate the possibility of its fast and accurate determination under the flow conditions.
      Graphical abstract image

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.025
      Issue No: Vol. 544 (2017)
  • A multi-substrate assay for finding physiologically effective inhibitors
           of myeloperoxidase
    • Authors: Louisa V. Forbes; Anthony J. Kettle
      Pages: 13 - 21
      Abstract: Publication date: 1 March 2018
      Source:Analytical Biochemistry, Volume 544
      Author(s): Louisa V. Forbes, Anthony J. Kettle
      Myeloperoxidase, an abundant neutrophil enzyme, promotes oxidative damage during inflammation by generating hypohalous acids and free radicals. Currently, there are no selective drugs to inhibit its adverse activity. This short-coming is partly due to the lack of screening assays that mimic the complex enzymatic activities of myeloperoxidase in vivo. We have developed an assay for myeloperoxidase activity that includes its major physiological substrates - chloride, thiocyanate, tyrosine, and urate. The multi-substrate assay monitors bleaching of 5-thio-2-nitrobenzoic acid and measures total oxidant production when hydrogen peroxide activates the enzyme. Known suicide inhibitors and tight-binders tested positive in the assay, whereas compounds that merely convert myeloperoxidase to reducible enzyme intermediates were poor inhibitors. The new assay revealed that some aromatic compounds, including tryptamine, inhibit myeloperoxidase by binding reversibly to the enzyme. Our multi-substrate assay is selective for physiologically relevant inhibitors and has potential for identifying new classes of myeloperoxidase inhibitors.
      Graphical abstract image

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.022
      Issue No: Vol. 544 (2017)
  • A carbon nanotube-enhanced real-time immuno-PCR for ultrasensitive
           detection of AHTN in water
    • Authors: Xiaohan Zhang; Huisheng Zhuang
      Pages: 22 - 28
      Abstract: Publication date: 1 March 2018
      Source:Analytical Biochemistry, Volume 544
      Author(s): Xiaohan Zhang, Huisheng Zhuang
      Polycyclic musks (PCMs) in the aquatic environment have become an emerging environmental issue because of their potential risk. The most commonly used method for analysis of PCMs is gas chromatography-mass spectrometer (GC-MS) with different sample extractions, which are somewhat expensive to operate, laborious and complex. In this paper, a carbon nanotube-enhanced real time immuno-PCR was developed for ultrasensitive detection of AHTN in water for the first time. The SWCNTs were used to immobilize numerous amino-DNA and polyclonal antibody to form polyclonal antibody-CNTs-DNA conjugates, which were used as a signal-amplifier in the proposed immunoassay system. This proposed carbon nanotube enhanced real time immuno-PCR assay was used to determine AHTN in water samples ranging from 5 pg/L-0.1 ng/L; using sample size as low as 10 μL. This proposed carbon nanotube enhanced real time immuno-PCR is the most ultrasensitive one for determination of AHTN in water without pre-concentration or extractions; and it provide a potential way for ultra-trace AHTN detection in the aquatic environment.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.024
      Issue No: Vol. 544 (2017)
  • Development of a pan-rickettsial molecular diagnostic test based on
           recombinase polymerase amplification assay
    • Authors: Jonas Kissenkötter; Sören Hansen; Susanne Böhlken-Fascher; Olusegun George Ademowo; Oladapo Elijah Oyinloye; Adeleye Solomon Bakarey; Gerhard Dobler; Dennis Tappe; Pranav Patel; Claus-Peter Czerny; Ahmed Abd El Wahed
      Pages: 29 - 33
      Abstract: Publication date: 1 March 2018
      Source:Analytical Biochemistry, Volume 544
      Author(s): Jonas Kissenkötter, Sören Hansen, Susanne Böhlken-Fascher, Olusegun George Ademowo, Oladapo Elijah Oyinloye, Adeleye Solomon Bakarey, Gerhard Dobler, Dennis Tappe, Pranav Patel, Claus-Peter Czerny, Ahmed Abd El Wahed
      Rickettsioses are zoonotic vector-transmitted bacterial infections leading to flu-like symptoms and can progress to severe illness in humans. The gold standard for diagnosis of rickettsial infections is the indirect immunofluorescence assay, a serological method which is not suitable for pathogen identification during the acute phase of the disease. Therefore, several real-time PCR assays were developed. These assays are very sensitive, but require high-equipped laboratories and well-trained personnel. Hence, in this study, a rapid point-of-need detection method was developed to detect all Rickettsia species. The 23S and 16S rRNA genes were targeted to develop a recombinase polymerase amplification (RPA) assay. Both 23S and 16S_RPA assays required between seven to ten minutes to amplify and detect one or ten DNA molecules/reaction, respectively. The 16S_RPA assay detected all tested species, whereas the 23S_RPA assay identified only species of the spotted fever and transitional rickettsial groups. All results were compared with real-time PCR assays directed against the same rickettsial genes. The RPA assays are easy to handle and produced quicker results in comparison to real-time PCRs. Both RPA assays were implemented in a mobile suitcase laboratory to ease the use in rural areas. This method can help to provide rapid management of rickettsial infections.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.018
      Issue No: Vol. 544 (2017)
  • Generation and characterization of an anti-delta like ligand-4 Nanobody to
           induce non-productive angiogenesis
    • Authors: Rasoul Baharlou; Nader Tajik; Mahdi Habibi-Anbouhi; Mohammad Ali Shokrgozar; Amir-Hassan Zarnani; Fatemeh Shahhosseini; Mahdi Behdani
      Pages: 34 - 41
      Abstract: Publication date: 1 March 2018
      Source:Analytical Biochemistry, Volume 544
      Author(s): Rasoul Baharlou, Nader Tajik, Mahdi Habibi-Anbouhi, Mohammad Ali Shokrgozar, Amir-Hassan Zarnani, Fatemeh Shahhosseini, Mahdi Behdani
      Antibody-based targeting of angiogenesis is a key approach for cancer treatment. Delta-like ligand 4 (DLL4) plays a pivotal role in tumor neovascular development and angiogenesis during tumor progression. It forecasts the prognosis of human malignancies and blocking its signaling can help to inhibit neovascularization and tumor metastasis. Nanobodies are the smallest antigen-binding domains of heavy chain antibodies in camelidae. The aim of this study was to develop a Nanobody against DLL4 and apply binding and functional approaches to target it. In this work, a Nanobody library against human recombinant DLL4 was developed. After panning, the periplasmic-extract (PE) of individual colonies were screened through ELISA. The interactions between Nanobody and DLL4 were assessed using immunohistochemistry and FACS. The functional assessment was carried out via tube formation assay. We selected a Nanobody (3Nb3) with a high binding signal to DLL4, associated with a binding affinity of 3.6 nM. It was demonstrated that 3Nb3 binds to native DLL4 on the surface of MKN cells and gastric carcinoma tissue, and also inhibits the maturation of capillary-like structures in HUVECs. The results were indicative of the potential of Nanobody for DLL4 identification and can broaden the scope for development of cancer diagnosis and treatment techniques.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.014
      Issue No: Vol. 544 (2017)
  • Correlation of serum sialyl Tn antigen values determined by immunoassay
           and SRM based method
    • Authors: Miki Tanaka-Okamoto; Ken Hanzawa; Mikio Mukai; Hidenori Takahashi; Masayuki Ohue; Yasuhide Miyamoto
      Pages: 42 - 48
      Abstract: Publication date: 1 March 2018
      Source:Analytical Biochemistry, Volume 544
      Author(s): Miki Tanaka-Okamoto, Ken Hanzawa, Mikio Mukai, Hidenori Takahashi, Masayuki Ohue, Yasuhide Miyamoto
      We previously identified four glycan tumor marker candidates using a HPLC-based method. One candidate was sialyl Tn (STN), NeuAcα2-6-GalNAc. In this study, glycans were prepared from sera by hydrazine treatment followed by fluorescent labeling with aminopyridine. Pyridylaminated-STN levels of 147 gastric cancer, 85 pancreatic cancer and 10 cholangiocarcinoma patients together with 102 normal controls were accurately quantified using HPLC separation followed by selected reaction monitoring (SRM) assay, which used a stable isotope, tetradeuterium-labeled pyridylamino glycan as an internal standard. Additionally, STN values were also quantified using conventional competitive inhibition radioimmunoassay (RIA). The two STN levels determined by RIA and SRM gave a similar distribution pattern in sera. STN levels were increased in sera from cancer patients compared to those from normal controls. Moreover, the STN levels in sera of cancer patients determined by the two different assay procedures showed a good correlation (i.e., correlation coefficient >0.9). Our results suggest it may be better to determine STN levels using SRM instead of RIA.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.026
      Issue No: Vol. 544 (2017)
  • Blocked recombinase polymerase amplification for mutation analysis of
           PIK3CA gene
    • Authors: Sara Martorell; Sarai Palanca; Ángel Maquieira; Luis A. Tortajada-Genaro
      Pages: 49 - 56
      Abstract: Publication date: 1 March 2018
      Source:Analytical Biochemistry, Volume 544
      Author(s): Sara Martorell, Sarai Palanca, Ángel Maquieira, Luis A. Tortajada-Genaro
      A blocked recombinase polymerase amplification (blocked-RPA) approach has been developed for the enrichment of mutated templates in heterogeneous specimens as tumor tissues. This isothermal amplification technique opens alternative solutions for meeting the technological demand of physician office laboratories. Herein, the detection of mutations in PIK3CA gene, such as p.E545K, and p.H1047L, is presented. The main element was an oligonucleotide (dideoxycytidine functionalized at 3′-end) which matched with wild-type sequence in the target locus. The amplification was performed operating at 37 °C during 40 min. The results demonstrated that the competition between the upstream primer and the blocker reduced the percentage of amplified wild-type allele, making the detection of the present mutation easier. For mutation discrimination, a fast hybridization assay was performed in microarray format on plastic chip and colorimetric detection. This approach enabled the reliable discrimination of specific mutations against a background of up to 95% wild-type DNA. The applicability of the method, based on the combination of blocked-RPA and low-cost chip hybridization, was successfully proven for the genotyping of various cancer cell lines as well as tumor tissues. The assignations agreed with those provided by next-generation sequencing. Therefore, these investigations would support a personalized approach to patient care based on the molecular signature of human cancers.
      Graphical abstract image

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.013
      Issue No: Vol. 544 (2017)
  • Fast and accurate enzyme activity measurements using a chip-based
           microfluidic calorimeter
    • Authors: Morten M.C.H. van Schie; Kourosh Honarmand Ebrahimi; Wilfred R. Hagen; Peter-Leon Hagedoorn
      Pages: 57 - 63
      Abstract: Publication date: 1 March 2018
      Source:Analytical Biochemistry, Volume 544
      Author(s): Morten M.C.H. van Schie, Kourosh Honarmand Ebrahimi, Wilfred R. Hagen, Peter-Leon Hagedoorn
      Recent developments in microfluidic and nanofluidic technologies have resulted in development of new chip-based microfluidic calorimeters with potential use in different fields. One application would be the accurate high-throughput measurement of enzyme activity. Calorimetry is a generic way to measure activity of enzymes, but unlike conventional calorimeters, chip-based calorimeters can be easily automated and implemented in high-throughput screening platforms. However, application of chip-based microfluidic calorimeters to measure enzyme activity has been limited due to problems associated with miniaturization such as incomplete mixing and a decrease in volumetric heat generated. To address these problems we introduced a calibration method and devised a convenient protocol for using a chip-based microfluidic calorimeter. Using the new calibration method, the progress curve of alkaline phosphatase, which has product inhibition for phosphate, measured by the calorimeter was the same as that recorded by UV-visible spectroscopy. Our results may enable use of current chip-based microfluidic calorimeters in a simple manner as a tool for high-throughput screening of enzyme activity with potential applications in drug discovery and enzyme engineering.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.028
      Issue No: Vol. 544 (2017)
  • Design and application of a fluorogenic assay for monitoring inflammatory
           caspase activity
    • Authors: Raj Ranganathan; Gena Lenti; Nicholas M. Tassone; Brian J. Scannell; Cathrine A. Southern; Caitlin E. Karver
      Pages: 1 - 7
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Raj Ranganathan, Gena Lenti, Nicholas M. Tassone, Brian J. Scannell, Cathrine A. Southern, Caitlin E. Karver
      Various fluorogenic assays exist for monitoring the activity of inflammatory caspases. However, there are no continuous assays that provide C-terminal substrate sequence specificity for inflammatory caspases. As a first step towards this, we have developed a continuous in vitro assay that relies on monitoring emission from tryptophan after cleavage of a quenching coumarin chromophore. The coumarin can be attached as an amino acid side chain or capping the C-terminus of the peptide. When the coumarin is a side chain, it allows for C-terminal and N-terminal sequence specificities to be explored. Using this assay, we obtained Michaelis-Menten kinetic data for four proof-of-principle peptides: WEHD-AMC (K M = 15 ± 2 μM), WEHD-MCA (K M = 93 ± 19 μM), WEHDG-MCA (K M = 21 ± 6 μM) and WEHDA-MCA (K M = 151 ± 37 μM), where AMC is 7-amino-4-methylcoumarin and MCA is β-(7-methoxy-coumarin-4-yl)-Ala. The results indicate the viability of this new assay approach in the design of effective fluorogenic substrates for inflammatory caspases.
      Graphical abstract image

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.11.023
      Issue No: Vol. 543 (2017)
  • Proteomic profiling of large myofibrillar proteins from dried and
           long-term stored polyacrylamide gels
    • Authors: Sandra Murphy; Kay Ohlendieck
      Pages: 8 - 11
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Sandra Murphy, Kay Ohlendieck
      A method for the utilization of dried polyacrylamide gels from the pre-proteomic era is described in order to enable the mass spectrometric analysis of long-term stored protein preparations. The in-gel digestion of high-molecular-mass proteins embedded in a 20-year old gel was carried out following gel re-swelling and resulted in the proteomic identification of a large number of proteins, including 3400 kDa titin, 800 kDa nebulin and myosin heavy chains of 220 kDa from rabbit skeletal muscle. These findings demonstrate that dried protein gels from past biochemical analyses can be successfully reused and analyzed by modern and refined mass spectrometric techniques.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.11.022
      Issue No: Vol. 543 (2017)
  • Voltammetric determination of thujone in herbal matrices in the presence
           of Triton X-100
    • Authors: Mateusz Kowalcze; Małgorzata Jakubowska
      Pages: 12 - 20
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Mateusz Kowalcze, Małgorzata Jakubowska
      A simple, sensitive and rapid method of low concentration of thujone determination using Controlled Growth Mercury Drop Electrode and Differential Pulse Voltammetry was described. The reduction of thujone in phosphoric acid or potassium nitrate, involves a quasi-reversible and adsorption-controlled two-electron process. In voltammetric experiments the addition of ethanol and Triton X–100 is recommended. Experimental conditions, such as composition of supporting electrolyte, concentration of Triton X-100 and operation parameters were established. The optimal electrolyte should contain: 0.04 mol L−1 phosphoric acid (pH = 1.9) or 0.05 mol L−1 potassium nitrate (pH = 6.4), 0.0002% Triton X-100 and 9.6% ethanol. The calibration graph was linear from 0.7 to 140 mgL−1 of thujone in H3PO4 as supporting electrolyte and from 0.7 to 115 mgL−1 of the analyte in KNO3. The detection limit was 0.4 mgL−1 in H3PO4 and 0.6 mgL−1 in KNO3, with the correlation coefficient of 0.998. The influence of interfering substances, such as Al(III), Bi(III), Cd(II), Cr(III), Cu(II), Pb(II), Tl(I), V(III) and Zn(II) was studied. The proposed method was validated by the thujone recovery study from the specially prepared spiked herbal matrices and alcohol extract. The considered real sample was thuja oil, in which thujone was successfully determined. All measurements were performed without sample preparation.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.11.024
      Issue No: Vol. 543 (2017)
  • Polymerization of hexamethylene diisocyanate in solution and a 260.23 m/z
           [M+H]+ ion in exposed human cells
    • Authors: Adam V. Wisnewski; Jian Liu; Carrie A. Redlich; Ala F. Nassar
      Pages: 21 - 29
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Adam V. Wisnewski, Jian Liu, Carrie A. Redlich, Ala F. Nassar
      Hexamethylene diisocyanate (HDI) is an important industrial chemical that can cause asthma, however pathogenic mechanisms remain unclear. Upon entry into the respiratory tract, HDI's N=C=O groups may undergo nucleophilic addition (conjugate) to host molecules (e.g. proteins), or instead react with water (hydrolyze), releasing CO2 and leaving a primary amine in place of the original N=C=O. We hypothesized that (primary amine groups present on) hydrolyzed or partially hydrolyzed HDI may compete with proteins and water as a reaction target for HDI in solution, resulting in polymers that could be identified and characterized using LC-MS and LC-MS/MS. Analysis of the reaction products formed when HDI was mixed with a pH buffered, isotonic, protein containing solution identified multiple [M+H]+ ions with m/z's and collision-induced dissociation (CID) fragmentation patterns consistent with those expected for dimers (259.25/285.23 m/z), and trimers (401.36/427.35 m/z) of partially hydrolyzed HDI (e.g. ureas/oligoureas). Human peripheral blood mononuclear cells (PBMCs) and monocyte-like U937, but not airway epithelial NCI-H292 cell lines cultured with these HDI ureas contained a novel 260.23 m/z [M+H]+ ion. LC-MS/MS analysis of the 260.23 m/z [M+H]+ ion suggest the formula C13H29N3O2 and a structure containing partially hydrolyzed HDI, however definitive characterization will require further orthogonal analyses.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.11.017
      Issue No: Vol. 543 (2017)
  • ImageJ-based semiautomatic method to analyze senescence in cell culture
    • Authors: Javier Lozano-Gerona; Ángel-Luis García-Otín
      Pages: 30 - 32
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Javier Lozano-Gerona, Ángel-Luis García-Otín
      β-Galactosidase accumulates in the lysosomes of senescent cells of certain tissues. Cell staining with X-gal is a common procedure to detect senescent cells in culture. However, the organelle nature of the staining makes automatic count impossible, requiring time-consuming manual counting or expensive alternative techniques such as flow cytometry to effectively determine the amount of stained cells. Here we present an analysis strategy for images of X-gal stained cells which can be implemented into a macro for the ImageJ software overcoming some of the drawbacks of computational analysis of organelle staining.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.11.020
      Issue No: Vol. 543 (2017)
  • Microplate chemiluminescent assay for HBV DNA detection using
           3-(10′-phenothiazinyl)propionic acid/N-morpholinopyridine pair as
           enhancer of HRP-catalyzed chemiluminescence
    • Authors: Oleg L. Bodulev; Anastasia V. Gribas; Ivan Yu. Sakharov
      Pages: 33 - 36
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Oleg L. Bodulev, Anastasia V. Gribas, Ivan Yu. Sakharov
      A sensitive sandwich assay for hepatitis B virus (HBV) DNA detection based on use of commercial CL-ELISA microplates was developed. To reveal the target the covalent conjugate of reporter oligonucleotide and horseradish peroxidase (HRP) was synthesized. An employment of enhanced chemiluminescence reaction, where 3-(10′-phenothiazinyl)propionic acid/N-morpholinopyridine pair was used as enhancer of HRP-catalyzed chemiluminescence, permitted to measure the enzyme activity of the conjugate with high sensitivity. Under the favorable conditions the limit of detection and a linear range of the assay were 3 pM and 0.07–2.0 nM, respectively. The coefficient of variation (CV) for determination of HBV DNA concentrations within the working range was lower than 4%. The obtained results demonstrated that the developed assay had high sensitivity and precision.
      Graphical abstract image

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.11.026
      Issue No: Vol. 543 (2017)
  • Evaluation of digestion methods for analysis of trace metals in mammalian
           tissues and NIST 1577c
    • Authors: Grace A. Binder; Rainer Metcalf; Zachary Atlas; Kenyon G. Daniel
      Pages: 37 - 42
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Grace A. Binder, Rainer Metcalf, Zachary Atlas, Kenyon G. Daniel
      Digestion techniques for ICP analysis have been poorly studied for biological samples. This report describes an optimized method for analysis of trace metals that can be used across a variety of sample types. Digestion methods were tested and optimized with the analysis of trace metals in cancerous as compared to normal tissue as the end goal. Anthropological, forensic, oncological and environmental research groups can employ this method reasonably cheaply and safely whilst still being able to compare between laboratories. We examined combined HNO3 and H2O2 digestion at 170 °C for human, porcine and bovine samples whether they are frozen, fresh or lyophilized powder. Little discrepancy is found between microwave digestion and PFA Teflon pressure vessels. The elements of interest (Cu, Zn, Fe and Ni) yielded consistently higher and more accurate values on standard reference material than samples heated to 75 °C or samples that utilized HNO3 alone. Use of H2SO4 does not improve homogeneity of the sample and lowers precision during ICP analysis. High temperature digestions (>165 °C) using a combination of HNO3 and H2O2 as outlined are proposed as a standard technique for all mammalian tissues, specifically, human tissues and yield greater than 300% higher values than samples digested at 75 °C regardless of the acid or acid combinations used. The proposed standardized technique is designed to accurately quantify potential discrepancies in metal loads between cancerous and healthy tissues and applies to numerous tissue studies requiring quick, effective and safe digestions.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.11.016
      Issue No: Vol. 543 (2017)
  • Use of alternative alkali chlorides in RT and PCR of polynucleotides
           containing G quadruplex structures
    • Authors: Fabiola Ramos-Alemán; Eva González-Jasso; Reynaldo C. Pless
      Pages: 43 - 50
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Fabiola Ramos-Alemán, Eva González-Jasso, Reynaldo C. Pless
      Several alkali chlorides were compared for their use in reverse transcription (RT) and PCR of different types of nucleic acid templates. On a test region of biological DNA incapable of forming G quadruplex (G4) structures, Taq DNA polymerase showed similar PCR performance with 50 mM KCl, CsCl, LiCl, and NaCl. In contrast, on a synthetic model polydeoxyribonucleotide prone to G4 formation, good PCR amplification was obtained with 50 mM CsCl, but little or none with LiCl or KCl. Similarly, in RT of a G4-prone model polyribonucleotide, MMLV reverse transcriptase produced a good yield with 50 mM CsCl, mediocre yields with LiCl or without added alkali chloride, and a poor yield with 50 mM KCl. The full RT-PCR assay starting from the G4-prone polyribonucleotide, showed good results with CsCl in both stages, poor results with LiCl, and no product formation with KCl. The model polynucleotides showed fast G quadruplex formation under PCR or RT conditions with 50 mM KCl, but not with CsCl or LiCl. The results argue for the use of CsCl instead of KCl for RT and PCR of G4-prone sequences. No advantage was observed when using the 7-deaza type nucleotide analog c7dGTP in PCR amplification of the G4-prone polydeoxyribonucleotide.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.11.021
      Issue No: Vol. 543 (2017)
  • Rapid profiling method for mammalian feces short chain fatty acids by
    • Authors: Takeshi Furuhashi; Kuniyo Sugitate; Takashi Nakai; Yusuke Jikumaru; Genki Ishihara
      Pages: 51 - 54
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Takeshi Furuhashi, Kuniyo Sugitate, Takashi Nakai, Yusuke Jikumaru, Genki Ishihara
      Short chain fatty acids (SCFAs) are key feces metabolites generated by gut bacteria fermentation. Despite the importance of profiling feces SCFAs, technical difficulties in analysis remain due to their volatility and hydrophilicity. We improve previous protocols to profile SCFAs and optimize the metabolite profiling platform for mammalian feces samples. In this study, we investigated feces as biological samples using gas chromatography-mass spectrometry (GC-MS). Isobutyl chloroformate was used for a derivatization in aqueous solution without drying out the samples. Ultimately, we envisage being able to determine the way in which gut bacteria fermentation influences host gut condition by using our rapid metabolite profiling methods.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.001
      Issue No: Vol. 543 (2017)
  • Development of a borreliosis assay: Mannan coated polyethylene sinter
           bodies as a new platform technology
    • Authors: Mohammed Alasel; Nina Dassinger; Doru Vornicescu; Michael Keusgen
      Pages: 55 - 61
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Mohammed Alasel, Nina Dassinger, Doru Vornicescu, Michael Keusgen
      Rapid diagnosis of Lyme borreliosis has been carried out on chemically modified porous polyethylene sinter bodies. Photografting of 2-propenol on sinter body's surface was performed as a first step, introducing active hydroxyl groups as a result of polyalcohol formation. The hydroxyl groups were used for further immobilization and could be linked via 3-aminopropyltriethoxysilane (APTES) to polysaccharides like mannan. Prone to coupling, mannan was activated using N, N'-disuccinimidyl carbonate (DSC) to allow smooth reaction with the primary amine groups of the silane layer. In a final preparation step, a recombinant fusion protein consisting of the mannan-binding domain of the lectin Concanavalin A (ConA) and a specific Borrelia surface antigen was immobilized by self-organization on the mannan surface. The fusion protein was used as biological interface structure. This strategy is highly efficient and resulted in a defined orientation of the antigen part of the fusion protein. Rapid and convenient differentiation could be then established between Borrelia-negative and a -positive serum even in 1000-fold diluted samples and detection of Lyme borreliosis in a rather early stage is likely. Furthermore, this generic strategy can be easily transferred to other bacterial or viral antigen structures.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.002
      Issue No: Vol. 543 (2017)
  • Exploring three different expression systems for recombinant expression of
           globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda
    • Authors: An Bracke; David Hoogewijs; Sylvia Dewilde
      Pages: 62 - 70
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): An Bracke, David Hoogewijs, Sylvia Dewilde
      Globins are among the best investigated proteins in biological and medical sciences and represent a prime tool for the study of the evolution of genes and the structure-function relationship of proteins. Here, we explore the recombinant expression of globins in three different expression systems: Escherichia coli, Pichia pastoris and the baculovirus infected Spodoptera frugiperda. We expressed two different human globin types in these three expression systems: I) the well-characterized neuroglobin and II) the uncharacterized, circular permutated globin domain of the large chimeric globin androglobin. It is clear from the literature that E.coli is the most used expression system for expression and purification of recombinant globins. However, the major disadvantage of E. coli is the formation of insoluble aggregates. We experienced that, for more complex multi-domain globins, like the chimeric globin androglobin, it is recommended to switch to a higher eukaryotic expression system.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.11.027
      Issue No: Vol. 543 (2017)
  • Highly specific real-time quantification of diverse microRNAs in human
           samples using universal primer set frame
    • Authors: Wancun Zhang; Jiaqi Zhang; Qi Zhang; Fang Hu; Yueqing Gu
      Pages: 71 - 78
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Wancun Zhang, Jiaqi Zhang, Qi Zhang, Fang Hu, Yueqing Gu
      In this study, one group of universal primer set frame, composed by one reverse transcription (RT) primer frame and a pair of quantitative real-time polymerase chain reaction (qRT-PCR) primer frames, was elaborately screened and designed by homebuilt software for sensitive and specific quantification of diverse miRNAs. The universal primer set frame can be applied for multiplex miRNAs detection by simply changing the RT-X part of RT primer frame and RP-Y part of qRT-PCR reverse primer frame based on target sequence. The maximum similarity of RT-Y, RT-Z and qRT-PCR forward primer to the human genome and human transcriptome is less than 76%, ensuring the high specificity in human sample detection. The high sensitivity and broad dynamic linear range of the developed approaches by using designed primer set frame were demonstrated on the in vitro detection of miR-21 and miR-155, with dynamic range of 10 fM to 10 nM and detection limit of 3.74 × 10−15 M and 5.81 × 10−15 M for miR-21 and miR-155, respectively. In particular, the developed assays also have high sequence specificity which could clearly discriminate a single base difference in miRNA sequence. The contents of miR-21 and miR-155 in tissue and serum samples have been successfully detected using the developed assays. Results indicated that miR-21 and miR-155 were elevated in cancer tissue and serum specimens than that of normal samples, implying the developed assays hold a great promise for further application in biomedical research and early clinical diagnosis. More importantly, the primer set frame can be universally used in any miRNA investigation.
      Graphical abstract image

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.004
      Issue No: Vol. 543 (2017)
  • Detection of simultaneous multi-mutations using base-quenched probe
    • Authors: Huihui Mao; Guanghua Luo; Jun Zhang; Ning Xu
      Pages: 79 - 81
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Huihui Mao, Guanghua Luo, Jun Zhang, Ning Xu
      The base-quenched probe method for detecting single nucleotide polymorphisms (SNPs) relies on real-time PCR and melting-curve approaches. Here, we applied the most common commercial fluorophores including FAM, HEX, CY5, CY3, TET, JOE, Texas Red and ROX for labeling probes to detect multi-mutations simultaneously according to the different fluorescence channels. Accuracy of the method was confirmed by direct sequencing. The results demonstrated that all above dyes could be influenced by bases and could be applied to detect SNPs. Furthermore, this method was applied to detect APOM rs707921, APOM rs707922 and MCP-1 rs1024611 simultaneously, which was demonstrated successfully.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.011
      Issue No: Vol. 543 (2017)
  • Validated MALDI-TOF-MS method for anthrax lethal factor provides early
           diagnosis and evaluation of therapeutics
    • Authors: Maribel Gallegos-Candela; Anne E. Boyer; Adrian R. Woolfitt; Judy Brumlow; Renato C. Lins; Conrad P. Quinn; Alex R. Hoffmaster; Gabriel Meister; John R. Barr
      Pages: 97 - 107
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Maribel Gallegos-Candela, Anne E. Boyer, Adrian R. Woolfitt, Judy Brumlow, Renato C. Lins, Conrad P. Quinn, Alex R. Hoffmaster, Gabriel Meister, John R. Barr
      Anthrax lethal factor (LF) is a zinc-dependent endoprotease and a critical virulence factor for Bacillus anthracis, the causative agent of anthrax. The mass spectrometry (MS) method for total-LF quantification includes three steps; 1) LF specific antibody capture/concentration, 2) LF-specific hydrolysis of a peptide substrate, and 3) detection and quantification of LF-cleaved peptides by isotope-dilution MALDI-TOF/MS. Recombinant LF spiked plasma was used for calibration and quality control (QC) materials. Specificity was 100% from analysis of serum and plasma from 383 non-infected humans, 31 rabbits, and 24 rhesus macaques. Sensitivity was 100% from 32 human clinical anthrax cases including infections by inhalation, ingestion, cutaneous and injection exposures and experimental infections for 29 rabbits and 24 rhesus macaques with inhalation anthrax. Robustness evaluation included sample storage, serum and plasma, antimicrobial and antitoxin effects and long-term performance. Data from 100 independent runs gave detection limits 0.01 ng/mL (111 amol/mL) for the 4-h method and 0.0027 ng/mL (30 amol/mL) for an alternate 20-h method. QC precision ranged from 7.7 to 14.8% coefficient of variation and accuracy from 0.2 to 9.8% error. The validated LF MS method provides sensitive quantification of anthrax total-LF using a robust high throughput platform for early diagnosis and evaluation of therapeutics during an anthrax emergency.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.007
      Issue No: Vol. 543 (2017)
  • A macrodomain-linked immunosorbent assay (MLISA) for
    • Authors: Jingwen Chen; Albert T. Lam; Yong Zhang
      Pages: 132 - 139
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Jingwen Chen, Albert T. Lam, Yong Zhang
      ADP-ribosyltransferases (ARTs) catalyze reversible additions of mono- and poly-ADP-ribose onto diverse types of proteins by using nicotinamide adenine dinucleotide (NAD+) as a cosubstrate. In the human ART superfamily, 14 out of 20 members are shown to catalyze endogenous protein mono-ADP-ribosylation and play important roles in regulating various physiological and pathophysiological processes. Identification of new modulators of mono-ARTs can thus potentially lead to discovery of novel therapeutics. In this study, we developed a macrodomain-linked immunosorbent assay (MLISA) for characterizing mono-ARTs. Recombinant macrodomain 2 from poly-ADP-ribose polymerase 14 (PARP14) was generated with a C-terminal human influenza hemagglutinin (HA) tag for detecting mono-ADP-ribosylated proteins. Coupled with an anti-HA secondary antibody, the generated HA-tagged macrodomain 2 reveals high specificity for mono-ADP-ribosylation catalyzed by distinct mono-ARTs. Kinetic parameters of PARP15-catalyzed automodification were determined by MLISA and are in good agreement with previous studies. Eight commonly used chemical tools for PARPs were examined by MLISA with PARP15 and PARP14 in 96-well plates and exhibited moderate inhibitory activities for PARP15, consistent with published reports. These results demonstrate that MLISA provides a new and convenient method for quantitative characterization of mono-ART enzymes and may allow identification of potent mono-ART inhibitors in a high-throughput-compatible manner.
      Graphical abstract image

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.019
      Issue No: Vol. 543 (2017)
  • Improved HPLC-method for estimation and correction of amino acid losses
           during hydrolysis of unknown samples
    • Authors: Anne Lamp; Martin Kaltschmitt; Oliver Lüdtke
      Pages: 140 - 145
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Anne Lamp, Martin Kaltschmitt, Oliver Lüdtke
      Amino acid analysis, commonly done by acid hydrolysis of proteins and HPLC analysis, faces one major problem: incomplete hydrolysis of stable amino acids and degradation of unstable amino acids are causing amino acid losses. As a result, amino acid recovery of unknown samples cannot be estimated. Some methods have been reported for correction of these factors in the past. This paper shows an improved and integrated method to overcome this problem by using stillage as an exemplary unknown sample material. Amino acid recovery from an unknown sample can be estimated by standard addition of a known protein. If the sample does not cause matrix effects during amino acid hydrolysis, recoveries of the standard protein are transferable to the sample. If the sample does cause matrix effects correction of amino acid losses can instead be done by determination of hydrolysis kinetics. Therefore, first order kinetics were used for amino acids that undergo degradation during hydrolysis. For all stable amino acids higher order kinetics were used, a novel approach to determine hydrolysis kinetics. The presented method can be a helpful tool for scientists who want to optimize amino acid analysis of a particular biomass substrate.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.009
      Issue No: Vol. 543 (2017)
  • Method validation in quantitative analysis of phase I and phase II
           metabolites of mitragynine in human urine using liquid
           chromatography-tandem mass spectrometry
    • Authors: Mei Jin Lee; Surash Ramanathan; Sharif Mahsufi Mansor; Keng Yoon Yeong; Soo Choon Tan
      Pages: 146 - 161
      Abstract: Publication date: 15 February 2018
      Source:Analytical Biochemistry, Volume 543
      Author(s): Mei Jin Lee, Surash Ramanathan, Sharif Mahsufi Mansor, Keng Yoon Yeong, Soo Choon Tan
      A method using solid phase extraction and liquid chromatography-tandem mass spectrometry to quantitatively detect mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine in human urine samples was developed and validated. The relevant metabolites were identified using multiple reaction monitoring in positive ionization mode using nalorphine as an internal standard. The method was validated for accuracy, precision, recovery, linearity, and lower limit of quantitation. The intra- and inter-day accuracy and precision were found in the range of 83.6–117.5% with coefficient of variation less than 13%. The percentage of recovery for mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine was within the range of 80.1–118.9%. The lower limit of quantification was 1 ng/mL for mitragynine, 2 ng/mL for 16-carboxy mitragynine, and 50 ng/mL for 9-O-demethyl mitragynine. The developed method was reproducible, high precision and accuracy with good linearity and recovery for mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine in human urine.

      PubDate: 2017-12-24T18:04:02Z
      DOI: 10.1016/j.ab.2017.12.021
      Issue No: Vol. 543 (2017)
  • Graphene paper supported MoS2 nanocrystals monolayer with Cu
           submicron-buds: High-performance flexible platform for sensing in sweat
    • Authors: Zhengyun Wang; Shuang Dong; Mengxi Gui; Muhammad Asif; Wei Wang; Feng Wang; Hongfang Liu
      Abstract: Publication date: Available online 9 December 2017
      Source:Analytical Biochemistry
      Author(s): Zhengyun Wang, Shuang Dong, Mengxi Gui, Muhammad Asif, Wei Wang, Feng Wang, Hongfang Liu
      Flexible sweat biosensors are of considerable current interest for the development of wearable smart miniature devices. In this work, we report a novel type of flexible and electrochemical sweat platform fabricated by depositing Cu submicron buds on freestanding graphene paper (GP) carrying MoS2 nanocrystals monolayer for bio-functional detection of glucose and lactate. Quantitative analysis of glucose and lactate was carried out by using amperometric i–t method. Linear ranges were obtained between 5 and 1775 μM for glucose and 0.01–18.4 mM for lactate, and their corresponding limits of detection were 500 nM and 0.1 μM, respectively. The platform demonstrates fast response, good selectivity, superb reproducibility and outstanding flexibility, which enable its use for monitoring glucose and lactate in human perspiration. The strategy of structurally integrating 3D transition metal, 0D transition metal sulfide and 2D graphene will provide new insight into the design of flexible electrodes for sweat glucose and lactate monitoring and a wider range of applications in biosensing, bioelectronics, and lab-on-a-chip devices.

      PubDate: 2017-12-12T13:00:34Z
      DOI: 10.1016/j.ab.2017.12.010
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