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Showing 1 - 200 of 3043 Journals sorted alphabetically
AASRI Procedia     Open Access   (Followers: 15)
Academic Pediatrics     Hybrid Journal   (Followers: 22, SJR: 1.402, h-index: 51)
Academic Radiology     Hybrid Journal   (Followers: 21, SJR: 1.008, h-index: 75)
Accident Analysis & Prevention     Partially Free   (Followers: 84, 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: 350, SJR: 0.726, h-index: 43)
Acta Automatica Sinica     Full-text available via subscription   (Followers: 3)
Acta Biomaterialia     Hybrid Journal   (Followers: 25, 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   (SJR: 0.123, h-index: 8)
Acta Histochemica     Hybrid Journal   (Followers: 3, SJR: 0.604, h-index: 38)
Acta Materialia     Hybrid Journal   (Followers: 240, 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: 23, 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)
Actas Urológicas Españolas (English Edition)     Full-text available via subscription   (Followers: 2)
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: 4)
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: 5)
Additive Manufacturing     Hybrid Journal   (Followers: 7, SJR: 1.039, h-index: 5)
Additives for Polymers     Full-text available via subscription   (Followers: 21)
Advanced Drug Delivery Reviews     Hybrid Journal   (Followers: 135, 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: 25, SJR: 0.169, h-index: 4)
Advances in Antiviral Drug Design     Full-text available via subscription   (Followers: 3)
Advances in Applied Mathematics     Full-text available via subscription   (Followers: 6, SJR: 1.054, h-index: 35)
Advances in Applied Mechanics     Full-text available via subscription   (Followers: 11, SJR: 0.801, h-index: 26)
Advances in Applied Microbiology     Full-text available via subscription   (Followers: 22, 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: 25, 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: 5, SJR: 2.139, h-index: 42)
Advances in Cellular and Molecular Biology of Membranes and Organelles     Full-text available via subscription   (Followers: 12)
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: 11)
Advances in Digestive Medicine     Open Access   (Followers: 6)
Advances in DNA Sequence-Specific Agents     Full-text available via subscription   (Followers: 5)
Advances in Drug Research     Full-text available via subscription   (Followers: 22)
Advances in Ecological Research     Full-text available via subscription   (Followers: 41, SJR: 3.25, h-index: 43)
Advances in Engineering Software     Hybrid Journal   (Followers: 25, SJR: 0.486, h-index: 10)
Advances in Experimental Biology     Full-text available via subscription   (Followers: 7)
Advances in Experimental Social Psychology     Full-text available via subscription   (Followers: 41, 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: 50, 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: 15, 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 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: 35, 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 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: 9)
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: 5)
Advances in Microbial Physiology     Full-text available via subscription   (Followers: 4, SJR: 1.44, h-index: 51)
Advances in Molecular and Cell Biology     Full-text available via subscription   (Followers: 22)
Advances in Molecular and Cellular Endocrinology     Full-text available via subscription   (Followers: 10)
Advances in Molecular Toxicology     Full-text available via subscription   (Followers: 7, 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: 3)
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: 15, 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: 8)
Advances in Plant Pathology     Full-text available via subscription   (Followers: 5)
Advances in Porous Media     Full-text available via subscription   (Followers: 4)
Advances in Protein Chemistry     Full-text available via subscription   (Followers: 17)
Advances in Protein Chemistry and Structural Biology     Full-text available via subscription   (Followers: 20, SJR: 1.5, h-index: 62)
Advances in Psychology     Full-text available via subscription   (Followers: 61)
Advances in Quantum Chemistry     Full-text available via subscription   (Followers: 5, 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 Research     Full-text available via subscription   (Followers: 353, 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: 7, SJR: 0.823, h-index: 27)
Advances in the Study of Behavior     Full-text available via subscription   (Followers: 30, SJR: 1.321, h-index: 56)
Advances in Veterinary Medicine     Full-text available via subscription   (Followers: 17)
Advances in Veterinary Science and Comparative Medicine     Full-text available via subscription   (Followers: 13)
Advances in Virus Research     Full-text available via subscription   (Followers: 5, SJR: 1.878, h-index: 68)
Advances in Water Resources     Hybrid Journal   (Followers: 43, 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: 325, 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: 5, SJR: 0.344, h-index: 6)
Ageing Research Reviews     Hybrid Journal   (Followers: 8, SJR: 3.289, h-index: 78)
Aggression and Violent Behavior     Hybrid Journal   (Followers: 405, 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: 30, SJR: 1.275, h-index: 74)
Agricultural Water Management     Hybrid Journal   (Followers: 39, SJR: 1.546, h-index: 79)
Agriculture and Agricultural Science Procedia     Open Access  
Agriculture and Natural Resources     Open Access   (Followers: 1)
Agriculture, Ecosystems & Environment     Hybrid Journal   (Followers: 54, 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: 10, 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  
Algal Research     Partially Free   (Followers: 8, 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: 8, SJR: 0.158, h-index: 9)
Alzheimer's & Dementia     Hybrid Journal   (Followers: 48, SJR: 4.289, h-index: 64)
Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring     Open Access   (Followers: 6)
Alzheimer's & Dementia: Translational Research & Clinical Interventions     Open Access   (Followers: 5)
American Heart J.     Hybrid Journal   (Followers: 49, SJR: 3.157, h-index: 153)
American J. of Cardiology     Hybrid Journal   (Followers: 47, SJR: 2.063, h-index: 186)
American J. of Emergency Medicine     Hybrid Journal   (Followers: 39, SJR: 0.574, h-index: 65)
American J. of Geriatric Pharmacotherapy     Full-text available via subscription   (Followers: 8, SJR: 1.091, h-index: 45)
American J. of Geriatric Psychiatry     Hybrid Journal   (Followers: 15, SJR: 1.653, h-index: 93)
American J. of Human Genetics     Hybrid Journal   (Followers: 31, SJR: 8.769, h-index: 256)
American J. of Infection Control     Hybrid Journal   (Followers: 25, SJR: 1.259, h-index: 81)
American J. of Kidney Diseases     Hybrid Journal   (Followers: 32, 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: 237, SJR: 2.255, h-index: 171)
American J. of Ophthalmology     Hybrid Journal   (Followers: 57, SJR: 2.803, h-index: 148)
American J. of Ophthalmology Case Reports     Open Access   (Followers: 5)
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: 25, SJR: 0.59, h-index: 45)
American J. of Pathology     Hybrid Journal   (Followers: 26, SJR: 2.653, h-index: 228)
American J. of Preventive Medicine     Hybrid Journal   (Followers: 22, SJR: 2.764, h-index: 154)
American J. of Surgery     Hybrid Journal   (Followers: 34, 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: 5)
Anaerobe     Hybrid Journal   (Followers: 4, SJR: 1.066, h-index: 51)
Anaesthesia & Intensive Care Medicine     Full-text available via subscription   (Followers: 57, SJR: 0.124, h-index: 9)
Anaesthesia Critical Care & Pain Medicine     Full-text available via subscription   (Followers: 11)
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: 37, SJR: 1.548, h-index: 152)
Analytical Biochemistry     Hybrid Journal   (Followers: 166, 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: 11)
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: 160, SJR: 1.907, h-index: 126)
Animal Feed Science and Technology     Hybrid Journal   (Followers: 5, SJR: 1.151, h-index: 83)
Animal Reproduction Science     Hybrid Journal   (Followers: 5, SJR: 0.711, h-index: 78)
Annales d'Endocrinologie     Full-text available via subscription   (Followers: 1, SJR: 0.394, h-index: 30)
Annales d'Urologie     Full-text available via subscription  
Annales de Cardiologie et d'Angéiologie     Full-text available via subscription   (SJR: 0.177, h-index: 13)
Annales de Chirurgie de la Main et du Membre Supérieur     Full-text available via subscription  
Annales de Chirurgie Plastique Esthétique     Full-text available via subscription   (Followers: 2, SJR: 0.354, h-index: 22)
Annales de Chirurgie Vasculaire     Full-text available via subscription   (Followers: 1)

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Journal Cover Analytical Biochemistry
  [SJR: 0.725]   [H-I: 154]   [166 followers]  Follow
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0003-2697 - ISSN (Online) 1096-0309
   Published by Elsevier Homepage  [3043 journals]
  • Electrochemical detection of C-reactive protein using Copper nanoparticles
           and hybridization chain reaction amplifying signal
    • Authors: Junjun Zhang; Wenjuan Zhang; Jinjin Guo; Junchun Wang; Yuzhong Zhang
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Junjun Zhang, Wenjuan Zhang, Jinjin Guo, Junchun Wang, Yuzhong Zhang
      In this study, a sandwich-type electrochemical immunosensor for the detection of C-reactive protein (CRP) is described. In design, Copper nanoparticles (Cu NPs) were used for signal tag and hybridization chain reaction (HCR)amplified output signal. The immunosensor fabrication involved three steps: (i) primary antibodies (Ab1) were immobilized on the surface of gold nanoparticles (Au NPs); (ii) the sandwich-type structure formation contained “primary antibodies-antigen-secondary antibodies conjugated with primer (Ab2-S0)”; and (iii) long DNA concatemers intercalating amounts of Cu NPs was linked to the sandwich-type structure via hybridization reaction. Differential pulse voltammetry (DPV) was used to record the response signal of the immunosensor in phosphate-buffered saline (PBS). Under optimal conditions, the anodic peak currents of Cu NPs at the peak potential of about 0.08V(VS.SCE) were linear with the logarithm of CRP concentration in the range of 1.0 fg mL−1 to 100 ng mL−1 with a detection limit of 0.33 fg mL−1 (at signal/noise [S/N] = 3). In addition, the practical application of immunosensor was evaluated by analyzing CRP in real human serum samples, the recoveries obtained were within 95.3%–103.8%, indicating the immunosensor possessed potential application ability for practical disease diagnosis.

      PubDate: 2017-10-08T07:46:38Z
      DOI: 10.1016/j.ab.2017.09.017
      Issue No: Vol. 539 (2017)
  • A compartmentalized culture device for studying the axons of CNS neurons
    • Authors: Mei-Yun Cheng; Hsing-Hua Ho; Tsung-Kai Huang; Chih-Fan Chuang; Hui-Yu Chen; Hui-Wen Chung; Wan-Chong Leong; Wen-Cheng Yang; Chien-Chung Fu; Yun-Hsin Hsu; Yen-Chung Chang
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Mei-Yun Cheng, Hsing-Hua Ho, Tsung-Kai Huang, Chih-Fan Chuang, Hui-Yu Chen, Hui-Wen Chung, Wan-Chong Leong, Wen-Cheng Yang, Chien-Chung Fu, Yun-Hsin Hsu, Yen-Chung Chang
      We report here the development of a compartmentalized culture device that allows the spatial separation of the somatodendrites and axons of central nervous system (CNS) neurons. The device consists of two compartments separated by a septum constructed by attaching a porous polycarbonate track etch (PCTE) filter on top of a microchannel-filled polydimethylsiloxane (PDMS) membrane. The surface and microchannels of the septum are coated and filled, respectively, with materials that support neuron growth and neurite migration. When rat hippocampal neurons are cultured in the top compartment, axons are the only processes that can migrate through the septum to the bottom compartment. The axons in the bottom compartment can be studied directly in real-time or through immunofluorescence staining after fixation. Axons containing ∼3 μg protein can be isolated from each device for biochemical analyses. In addition, the septum also impedes the movement of small molecules between the top and bottom compartments. This feature allows the somatodendrites and axons of neurons, which occupy the top and bottom compartments of the device, respectively, to be manipulated independently. The potential applications of the device as a tool in diverse studies concerning neuronal axons and in screening reagents that regulate axonal functions have also been discussed.

      PubDate: 2017-10-08T07:46:38Z
      DOI: 10.1016/j.ab.2017.09.013
      Issue No: Vol. 539 (2017)
  • Preparation of wool follicles for proteomic studies
    • Authors: Jeffrey E. Plowman; Joy L. Woods; Bede van Schaijik; Duane P. Harland
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Jeffrey E. Plowman, Joy L. Woods, Bede van Schaijik, Duane P. Harland
      A variety of techniques were applied to wool follicles stored in William's E culture medium to optimise the extraction of keratin and keratin associated proteins (KAPs). A time course study indicated that the maximum storage time for live skin in this buffer at 20 °C was 24 h, after which degradative loss of protein became significant. Maceration of the skin for 10 min followed by reciprocal action shaking for 14 h had a detrimental effect on keratin extractability. The best approach involved using a Dounce homogeniser as this resulted in the highest amount of Type I and II keratins and KAPs.

      PubDate: 2017-10-08T07:46:38Z
      DOI: 10.1016/j.ab.2017.08.020
      Issue No: Vol. 539 (2017)
  • DNA interaction with platinum-based cytostatics revealed by DNA sequencing
    • Authors: Kristyna Smerkova; Tomas Vaculovic; Marketa Vaculovicova; Jindrich Kynicky; Martin Brtnicky; Tomas Eckschlager; Marie Stiborova; Jaromir Hubalek; Vojtech Adam
      Pages: 22 - 28
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Kristyna Smerkova, Tomas Vaculovic, Marketa Vaculovicova, Jindrich Kynicky, Martin Brtnicky, Tomas Eckschlager, Marie Stiborova, Jaromir Hubalek, Vojtech Adam
      The main mechanism of action of platinum-based cytostatic drugs – cisplatin, oxaliplatin and carboplatin – is the formation of DNA cross-links, which restricts the transcription due to the disability of DNA to enter the active site of the polymerase. The polymerase chain reaction (PCR) was employed as a simplified model of the amplification process in the cell nucleus. PCR with fluorescently labelled dideoxynucleotides commonly employed for DNA sequencing was used to monitor the effect of platinum-based cytostatics on DNA in terms of decrease in labeling efficiency dependent on a presence of the DNA-drug cross-link. It was found that significantly different amounts of the drugs - cisplatin (0.21 μg/mL), oxaliplatin (5.23 μg/mL), and carboplatin (71.11 μg/mL) - were required to cause the same quenching effect (50%) on the fluorescent labelling of 50 μg/mL of DNA. Moreover, it was found that even though the amounts of the drugs was applied to the reaction mixture differing by several orders of magnitude, the amount of incorporated platinum, quantified by inductively coupled plasma mass spectrometry, was in all cases at the level of tenths of μg per 5 μg of DNA.

      PubDate: 2017-10-14T13:38:05Z
      DOI: 10.1016/j.ab.2017.09.018
      Issue No: Vol. 539 (2017)
  • Isolation of a peptide from Ph.D.-C7C phage display library for detection
           of Cry1Ab
    • Authors: Yun Wang; Qian Wang; Ai-hua Wu; Zhen-ping Hao; Xian-jin Liu
      Pages: 29 - 32
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Yun Wang, Qian Wang, Ai-hua Wu, Zhen-ping Hao, Xian-jin Liu
      Traditional ELISA methods of using animal immunity yield antibodies for detection Cry toxin. Not only is this incredibly harmful to the animals, but is also time-intensive. Here we developed a simple method to yield the recognition element. Using a critical selection strategy and immunoassay we confirmed a clone from the Ph.D-C7C phage library, which has displayed the most interesting Cry1Ab-binding characteristics examined in this study (Fig. 1). The current study indicates that isolating peptide is an alternative method for the preparation of a recognition element, and that the developed assay is a potentially useful tool for detecting Cry1Ab.

      PubDate: 2017-10-14T13:38:05Z
      DOI: 10.1016/j.ab.2017.03.004
      Issue No: Vol. 539 (2017)
  • Rapid quantification of glutaminase 2 (GLS2)-related metabolites by
    • Authors: Guan-Yuan Chen; Hsi-Chun Chao; Hsiao-Wei Liao; I-Lin Tsai; Ching-Hua Kuo
      Pages: 39 - 44
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Guan-Yuan Chen, Hsi-Chun Chao, Hsiao-Wei Liao, I-Lin Tsai, Ching-Hua Kuo
      Glutamine, glutamate and glutathione are key modulators of excessive oxidative stress in tumor cells. In this study, we developed a rapid and accurate HILIC-MS/MS method to simultaneously determine concentrations of cellular glutamine, glutamate and glutathione. A bared silica HILIC column was employed to analyze these polar metabolites. The LC-MS parameters were optimized to achieve high sensitivity and selectivity. The analysis can be completed within 4 min under optimal conditions. The method was validated in terms of accuracy, precision, and linearity. Intra-day (n = 9) precision was within 2.68–6.24% among QCs. Inter-day precision (n = 3) was below 12.4%. The method accuracy was evaluated by the recovery test, and the accuracy for three analytes were between 91.6 and 110%. The developed method was applied to study antioxidant function of GLS2 in non-small cell lung cancer cells. Changes in concentrations of glutamine, glutamate and glutathione revealed that the overexpression of GLS2 could effectively decrease oxidative stress. In summary, this study developed a rapid HILIC-MS/MS method for quantification of GLS2-related metabolites that could facilitate elucidation of the role of GLS2 in tumor development.

      PubDate: 2017-10-14T13:38:05Z
      DOI: 10.1016/j.ab.2017.10.002
      Issue No: Vol. 539 (2017)
  • Method to minimize ozone effect on Cy5 fluorescent intensity in DNA
    • Authors: Youngjun Kim; Hyunhee Seo; Miseon Jeong; Kiheon Lee; Inho Lee; Kyeonga So; Mikyung Kim; Yookyung Lee; Seonah Kim; Taejin Kim
      Pages: 1 - 4
      Abstract: Publication date: 1 December 2017
      Source:Analytical Biochemistry, Volume 538
      Author(s): Youngjun Kim, Hyunhee Seo, Miseon Jeong, Kiheon Lee, Inho Lee, Kyeonga So, Mikyung Kim, Yookyung Lee, Seonah Kim, Taejin Kim
      Cyanine 5 (Cy5) is an established fluorescent dye in microarray analysis. It is degraded rapidly when exposed to atmospheric ozone during post-hybridization washes, which leads to loss of fluorescent intensity. To minimize this undesirable effect, we coated microarray slides with sodium dodecyl sulfate (SDS) solution at post-hybridization washes. The fluorescent intensities on coated slides were more stable than those on uncoated slide. We also performed the microarrays with SDS solution for a year to check the solution's effectiveness along with seasonal changes of atmospheric ozone level. Consistent results in microarray analysis were obtained using Cy5 dye under atmospheric ozone.

      PubDate: 2017-09-23T23:34:00Z
      DOI: 10.1016/j.ab.2017.09.001
      Issue No: Vol. 538 (2017)
  • Colorimetric detection of D-dimer in a paper-based immunodetection device
    • Authors: Sónia Ruivo; Ana M. Azevedo; Duarte M.F. Prazeres
      Pages: 5 - 12
      Abstract: Publication date: 1 December 2017
      Source:Analytical Biochemistry, Volume 538
      Author(s): Sónia Ruivo, Ana M. Azevedo, Duarte M.F. Prazeres
      A microfluidic paper-based analytical device (μPADs) immunoassay for detection of the blood native biomarker D-dimer is reported. The μPAD is created by wax printing on a single piece of chromatographic paper and combined with an anti-D-dimer capture antibody and conjugates of anti-D-dimer antibody with 40 nm gold nanoparticles. The presence of D-dimer in buffer/simulated plasma samples is successfully reported for concentrations as low as 15 ng D-dimer/mL. Linearity between signal intensity and D-dimer concentration is observed up to 100 ng/mL. Using an appropriate dilution, the test could be used to yield positive results only for those samples with a D-dimer concentration above the clinically relevant threshold range of 250–500 ng/mL. Finally, the merits and pitfalls of using μPADs as compared to lateral flow devices in immunoassays are discussed.

      PubDate: 2017-09-23T23:34:00Z
      DOI: 10.1016/j.ab.2017.09.009
      Issue No: Vol. 538 (2017)
  • Ultrasensitive and rapid immuno-detection of human IgE anti-therapeutic
           horse sera using an electrochemical immunosensor
    • Authors: Isis C. Prado; André L.A. Souza; David W. Provance-Jr; Ricardo J. Cassella; Salvatore G. De-Simone
      Pages: 13 - 19
      Abstract: Publication date: 1 December 2017
      Source:Analytical Biochemistry, Volume 538
      Author(s): Isis C. Prado, André L.A. Souza, David W. Provance-Jr, Ricardo J. Cassella, Salvatore G. De-Simone
      Antivenom allergy disease mediated by patient IgE is an important public health care concern. To improve detection of hypersensitive individuals prior to passive antibody therapy, an amperometric immunosensor was developed to detect reactive human IgE. Whole horse IgG3 (hoIgG3) was immobilized onto the surface of carbon or gold screen-printed electrodes through a cross-linking solution of glutaraldehyde on a chitosan film. Sera from persons with a known allergic response to hoIgG3 or non-allergic individuals was applied to the sensor. Bound human IgE (humIgE) was detected by an anti-humIgE antibody through a quantitative amperometric determination by tracking via the electrochemical reduction of the quinone generated from the hydroquinone with the application of a potential of 25 mV. The optimal immunosensor configuration detected reactive humIgE at a dilution of 1:1800 of the human sera that represent a detection limit of 0.5 pg/mL. Stability testing demonstrated that through 20 cycles of a scan, the specificity and performance remained robust. The new immunosensor successfully detected humIgE antibodies reactive against hoIgG3, which could allow the diagnosis of potential allergenic patients needing therapeutic antivenom preparations from a horse.

      PubDate: 2017-09-23T23:34:00Z
      DOI: 10.1016/j.ab.2017.09.008
      Issue No: Vol. 538 (2017)
  • Ultrasensitive microRNA-21 detection based on DNA hybridization chain
           reaction and SYBR Green dye
    • Authors: Zhi Li; Bingchen Li; Yunlei Zhou; Huanshun Yin; Jun Wang; Shiyun Ai
      Pages: 20 - 25
      Abstract: Publication date: 1 December 2017
      Source:Analytical Biochemistry, Volume 538
      Author(s): Zhi Li, Bingchen Li, Yunlei Zhou, Huanshun Yin, Jun Wang, Shiyun Ai
      It is extremely important for quantifying trace microRNAs in the biomedical applications. In this study, an ultrasensitive, rapid and efficient label-free fluorescence method was proposed and applied for detecting microRNA-21 in serum of gastric cancer patients based on DNA hybridization chain reaction (HCR). DNA H1 and DNA H2 were designed and used as hairpin probes, the HCR was proceeded in the presence of target microRNAs. Amounts of SYBR Green І dyes were used as signal molecules to intercalate long DNA concatemers from HCR, which guaranteed the model of label-free fluorescence and strong fluorescence density. The detection method showed a wide linear region from 1 fM to 105 fM, and the limit of detection was 0.2554 fM (at S/N = 3) for microRNAs. The results showed that this method had an excellent specificity and reproducibility. Furthermore, the label-free fluorescence strategy exhibited a sensitive response to microRNA-21 in real serum samples of gastric cancer patients and the results obtained were in accordance with reference method (R2 = 0.994). Overall, the proposed strategy could be satisfactory for rapid, ultrasensitive and efficient detection of microRNA-21, and held great potentials in clinic diagnosis of gastric cancer.
      Graphical abstract image

      PubDate: 2017-09-23T23:34:00Z
      DOI: 10.1016/j.ab.2017.09.007
      Issue No: Vol. 538 (2017)
  • Fluorescence and magnetic nanocomposite Fe3O4@SiO2@Au MNPs as peroxidase
           mimetics for glucose detection
    • Authors: Shajie Luo; Yaqin Liu; Hanbing Rao; Yanying Wang; Xianxiang Wang
      Pages: 26 - 33
      Abstract: Publication date: 1 December 2017
      Source:Analytical Biochemistry, Volume 538
      Author(s): Shajie Luo, Yaqin Liu, Hanbing Rao, Yanying Wang, Xianxiang Wang
      In this paper, multifunction nanoparticles (MNPs), Fe3O4@SiO2@Au MNPs, with properties of superparamagnetism, fluorescence and peroxidase-like catalytic activity were synthesized in the aqueous phase. The synthesized composites were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), Fourier translation infrared spectrum (FT-IR) and fluorometer. The results show that the multifunctional nanomaterials have good magnetic and fluorescence properties. Then, the mimetic properties of this material were investigated. The as-synthesized Fe3O4@SiO2@Au MNPs exhibited the best catalytic activity for peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) at the reaction temperature of 70 °C and pH of 3. Compared with free Fe3O4 MNPs and BSA-Au nanoclusters (NCs), the composites have better catalytic activity at higher temperature and lower pH, indicating that Fe3O4@SiO2@Au MNPs can work in more severe environment. In practical application, we have successfully established the colorimetric method for the detection of H2O2 and glucose with the detection range of 1 × 10−6 ∼ 4 × 10−5 M and 5 × 10−6 ∼ 3.5 × 10−4 M, and the detection limit of 6 × 10−7 M and 3.5 × 10−6 M, respectively. The method was also successfully applied in the detection of real samples. Furthermore, since the fluorescence of Fe3O4@SiO2@Au MNPs was quenched by H2O2, a method for the visual detection of glucose was established.
      Graphical abstract image

      PubDate: 2017-09-23T23:34:00Z
      DOI: 10.1016/j.ab.2017.09.006
      Issue No: Vol. 538 (2017)
  • Gas chromatography tandem mass spectrometry offers advantages for urinary
           steroids analysis
    • Authors: Natalie Homer; Sanjay Kothiya; Alison Rutter; Brian R. Walker; Ruth Andrew
      Pages: 34 - 37
      Abstract: Publication date: 1 December 2017
      Source:Analytical Biochemistry, Volume 538
      Author(s): Natalie Homer, Sanjay Kothiya, Alison Rutter, Brian R. Walker, Ruth Andrew
      Gas chromatography mass spectrometry has been the lynchpin of clinical assessment of steroid profiles for ∼3 decades. The improvements in assay performance offered by tandem mass spectrometry were assessed. Across the spectrum of glucocorticoid and androgen analytes tested, limits of detection and quantitation were ∼20 fold lower with triple than single quadrupole systems, but the more noticeable improvement was that signal to noise was substantially improved and the linear range wider. These benefits allowed more reliable and concomitant measurement of steroids with substantially different abundances and in smaller volumes of urine.

      PubDate: 2017-09-23T23:34:00Z
      DOI: 10.1016/j.ab.2017.09.002
      Issue No: Vol. 538 (2017)
  • Determination of protein thiolation index (PTI) as a biomarker of
           oxidative stress in human serum
    • Authors: Daniela Giustarini; Federico Galvagni; Graziano Colombo; Isabella Dalle-Donne; Aldo Milzani; Anna Maria Aloisi; Ranieri Rossi
      Pages: 38 - 41
      Abstract: Publication date: 1 December 2017
      Source:Analytical Biochemistry, Volume 538
      Author(s): Daniela Giustarini, Federico Galvagni, Graziano Colombo, Isabella Dalle-Donne, Aldo Milzani, Anna Maria Aloisi, Ranieri Rossi
      We have introduced protein thiolation index (PTI), i.e. the molar ratio of the sum of all low molecular mass thiols bound to plasma proteins to protein free cysteinyl residues, as a sensitive biomarker of oxidative stress. According to the original procedure its determination requires a rapid separation of plasma and a specific treatment of samples to stabilize thiols. Here we demonstrate that samples can be collected without use of any anticoagulant to prevent blood clotting and without any stabilization of thiols too. This simplification of the determination of PTI makes its analysis more feasible also in routine clinical laboratories.

      PubDate: 2017-09-23T23:34:00Z
      DOI: 10.1016/j.ab.2017.09.010
      Issue No: Vol. 538 (2017)
  • Menadione-mediated WST1 reduction assay for the determination of metabolic
           activity of cultured neural cells
    • Authors: Karsten Stapelfeldt; Eric Ehrke; Johann Steinmeier; Wiebke Rastedt; Ralf Dringen
      Pages: 42 - 52
      Abstract: Publication date: 1 December 2017
      Source:Analytical Biochemistry, Volume 538
      Author(s): Karsten Stapelfeldt, Eric Ehrke, Johann Steinmeier, Wiebke Rastedt, Ralf Dringen
      Cellular reduction of tetrazolium salts to their respective formazans is frequently used to determine the metabolic activity of cultured cells as an indicator of cell viability. For membrane-impermeable tetrazolium salts such as WST1 the application of a membrane-permeable electron cycler is usually required to mediate the transfer of intracellular electrons for extracellular WST1 reduction. Here we demonstrate that in addition to the commonly used electron cycler M-PMS, menadione can also serve as an efficient electron cycler for extracellular WST1 reduction in cultured neural cells. The increase in formazan absorbance in glial cell cultures for the WST1 reduction by menadione involves enzymatic menadione reduction and was twice that recorded for the cytosolic enzyme-independent WST1 reduction in the presence of M-PMS. The optimized WST1 reduction assay allowed within 30 min of incubation a highly reliable detection of compromised cell metabolism caused by 3-bromopyruvate and impaired membrane integrity caused by Triton X-100, with a sensitivity as good as that of spectrophotometric assays which determine cellular MTT reduction or lactate dehydrogenase release. The short incubation period of 30 min and the observed good sensitivity make this optimized menadione-mediated WST1 reduction assay a quick and reliable alternative to other viability and toxicity assays.

      PubDate: 2017-09-23T23:34:00Z
      DOI: 10.1016/j.ab.2017.09.011
      Issue No: Vol. 538 (2017)
  • A novel analytical procedure for assaying lysozyme activity using an
           end-blocked chitotetraose derivative as substrate
    • Authors: Makoto Ogata; Megumi Matsui; Haruka Kono; Yuka Matsuzaki; Yuna Kato; Taichi Usui
      Pages: 64 - 70
      Abstract: Publication date: 1 December 2017
      Source:Analytical Biochemistry, Volume 538
      Author(s): Makoto Ogata, Megumi Matsui, Haruka Kono, Yuka Matsuzaki, Yuna Kato, Taichi Usui
      An end-modified β-d-galactosyl chitotetraose derivative [44-O-β-d-galactosyl-β-tri-N-acetylchitotriosyl 2-acetamide-2,3-dideoxy-glucopyranose; Gal(GlcN)3D] was designed and synthesized from chitin tetrasaccharide. The derivative was chemically modified by dehydration of the reducing end GlcN and enzymatic addition of a Gal group to the non-reducing end GlcN. Hydrolysis of Gal(GlcN)3D and related compounds using hen egg-white lysozyme was then examined. Gal(GlcN)3D was specifically cleaved to Gal(GlcN)2 and GlcND. Kinetic studies and docking simulations were further conducted to elucidate its mode of binding to lysozyme. These analyses revealed the binding of Gal(GlcN)3D to lysozyme is more favorable than that of (GlcN)4D. We conclude the 4-O-substituted Gal group at the non-reducing end of Gal(GlcN)3D does not prohibit the action of lysozyme, but gives some affinity to the subsite (i.e. equivalent to GlcN). From these results, a new assay method for quantifying lysozyme was established by utilizing the Morgan-Elson reaction based on the generation of product D (2-acetamide-2,3-dideoxy-glucopyranose), which serves as a chromophore, formed from Gal(GlcN)3D by lysozyme through a conjugated reaction involving β-N-acetylhexosaminidase. The assay system gave a linear dose-response curve in the range of 2–31 μg of lysozyme during a 15 min incubation. This novel assay method for the quantification of lysozyme is highly specific, sensitive, accurate and reproducible.
      Graphical abstract image

      PubDate: 2017-10-08T07:46:38Z
      DOI: 10.1016/j.ab.2017.09.015
      Issue No: Vol. 538 (2017)
  • Capillary electrophoresis – Mass spectrometry metabolomics analysis
           revealed enrichment of hypotaurine in rat glioma tissues
    • Authors: Peng Gao; Min Ji; Xueyan Fang; Yingyang Liu; Zhigang Yu; Yunfeng Cao; Aijun Sun; Liang Zhao; Yong Zhang
      Pages: 1 - 7
      Abstract: Publication date: 15 November 2017
      Source:Analytical Biochemistry, Volume 537
      Author(s): Peng Gao, Min Ji, Xueyan Fang, Yingyang Liu, Zhigang Yu, Yunfeng Cao, Aijun Sun, Liang Zhao, Yong Zhang
      Glioma is one of the most lethal brain malignancies with unknown etiologies. Many metabolomics analysis aiming at diverse kinds of samples had been performed. Due to the varied adopted analytical platforms, the reported disease-related metabolites were not consistent across different studies. Comparable metabolomics results are more likely to be acquired by analyzing the same sample types with identical analytical platform. For tumor researches, tissue samples metabolomics analysis own the unique advantage that it can gain more direct insight into disease-specific pathological molecules. In this light, a previous reported capillary electrophoresis – mass spectrometry human tissues metabolomics analysis method was employed to profile the metabolome of rat C6 cell implantation gliomas and the corresponding precancerous tissues. It was found that 9 metabolites increased in the glioma tissues. Of them, hypotaurine was the only metabolite that enriched in the malignant tissues as what had been reported in the relevant human tissues metabolomics analysis. Furthermore, hypotaurine was also proved to inhibit α-ketoglutarate-dependent dioxygenases (2-KDDs) through immunocytochemistry staining and in vitro enzymatic activity assays by using C6 cell cultures. This study reinforced the previous conclusion that hypotaurine acted as a competitive inhibitor of 2-KDDs and proved the value of metabolomics in oncology studies.

      PubDate: 2017-09-05T19:57:42Z
      DOI: 10.1016/j.ab.2017.08.012
      Issue No: Vol. 537 (2017)
  • Measurement of O-GlcNAcylated endothelial nitric oxide synthase by using
           2′,5′-ADP-Sepharose pull-down assay
    • Authors: Yang Long; Jianghong Yan; Suxin Luo; Zhenguo Liu; Yong Xia
      Pages: 8 - 12
      Abstract: Publication date: 15 November 2017
      Source:Analytical Biochemistry, Volume 537
      Author(s): Yang Long, Jianghong Yan, Suxin Luo, Zhenguo Liu, Yong Xia
      Endothelial nitric oxide synthase (eNOS) plays central roles in cardiovascular regulation and disease. eNOS function is critically affected by O-linked N-acetylglucosamine (O-GlcNAc) modification. The present method for measuring O-GlcNAcylated eNOS relies on immunoprecipitation. Such method exhibits low detection efficiency and is also costly. We here report a simplified assay by employing the high binding affinity of eNOS with the 2′,5′-ADP-Sepharose resins. Together with the O-GlcNAc antibody, this assay readily allows the detection of O-GlcNAcylated eNOS in both cultured endothelial cells and rat vascular tissues. By using this assay, we demonstrate that eNOS O-GlcNAcylation is markedly elevated in the vessels of diabetic rats. Thus, a 2′,5′-ADP-Sepharose-based pull-down assay is developed to measure O-GlcNAcylated eNOS. This assay is simple and efficient in detecting O-GlcNAcylated eNOS in cultured cells and animal tissues under both normal and disease conditions.

      PubDate: 2017-09-05T19:57:42Z
      DOI: 10.1016/j.ab.2017.08.017
      Issue No: Vol. 537 (2017)
  • Imaged capillary isoelectric focusing in native condition: A novel and
           successful example
    • Authors: Xin Zhang; Letha Chemmalil; Julia Ding; Nesredin Mussa; Zhengjian Li
      Pages: 13 - 19
      Abstract: Publication date: 15 November 2017
      Source:Analytical Biochemistry, Volume 537
      Author(s): Xin Zhang, Letha Chemmalil, Julia Ding, Nesredin Mussa, Zhengjian Li
      Imaged capillary isoelectric focusing (icIEF) separates ampholytic components of biomolecules in an electric field according to their isoelectric points and has been used for protein charge variants quantification and characterization. Denaturants are ordinarily incorporated into icIEF to stabilize charge species in solution. In certain circumstances, however, denaturants are detrimental to stable isoelectric separation of proteins due to their unique structural and biophysical features, such as an aggregation-prone antibody we encountered recently. Here we report our novel matrix formula non-detergent sulfobetaine and taurine (NDSB-T). It is deprived of denaturants that notably ameliorates the assay robustness and peak resolution for this antibody. NDSB-T is a combination of non-detergent sulfobetaine (NDSB) and taurine possessing the stabilization and separation power while maintaining protein integrity. As a result, assay throughputs are tremendously increased for more than 10 folds along with extraordinarily improved assay accuracy. Furthermore, NDSB-T can separate and quantify protein charge species in native state and therefore avoid partial denaturation derived peaks which are often misleading and hard to characterize. NDSB-T may be a valuable tool for proteins incompatible with conventional icIEF matrices and potentially opens a new window for icIEF assay in native conditions.

      PubDate: 2017-09-30T12:02:11Z
      DOI: 10.1016/j.ab.2017.08.014
      Issue No: Vol. 537 (2017)
  • Isotope-dilution liquid chromatography-tandem mass spectrometry for
           sensitive quantification of human insulin in serum using
    • Authors: Yohei Sakaguchi; Tomoya Kinumi; Akiko Takatsu
      Pages: 26 - 32
      Abstract: Publication date: 15 November 2017
      Source:Analytical Biochemistry, Volume 537
      Author(s): Yohei Sakaguchi, Tomoya Kinumi, Akiko Takatsu
      An isotope-dilution mass spectrometry (IDMS) method for measuring insulin levels in human serum was developed using C-terminal-derivatization method coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The carboxyl groups of Glu-C-cleavage products were derivatized with 1-(2-pyrimidinyl)piperazine to increase MS/MS sensitivity and IDMS quantification, resulting in increases in LC-MS/MS peak areas of derivatized Glu-C-cleavage products of human insulin by ∼23-(A5–17 peptide) to 49-fold(B14–21 peptide), respectively, as compared with results observed in the absence of derivatization. Separation was achieved on a C18 column by gradient elution at 0.3 mL/min, with a mobile phase composed of 0.1% formic acid in acetonitrile and water. Validation studies of target peptides (B1–13 peptide and B14–21 peptide) revealed a linear response in the range of 0.05 ng/mL to 10 ng/mL (regression coefficient, r 2 = 0.9987 and 0.9988, respectively), a relative standard deviation within and between days of <8.6%, and spike and recovery test results indicating mean recoveries ranging from 100.2% to 106.6%. Comparison with an established commercial immunoassay showed high correlation (r 2 = 0.9943 and 0.9944, B1–13 peptide and B14–21 peptide, respectively) at serum concentrations of between 0.20 ng/mL and 1.51 ng/mL. These findings suggested that this IDMS-based approach was able to quantify human serum insulin with high sensitivity and precision in the reference interval and indicated a potential for determining serum-insulin reference-measurement procedures to allow traceable measurement.

      PubDate: 2017-09-05T19:57:42Z
      DOI: 10.1016/j.ab.2017.08.019
      Issue No: Vol. 537 (2017)
  • A generic approach for simultaneous measurements of total antibody and
           cleavable antibody-conjugated drug by LC/MS/MS
    • Authors: Ling Xu; Laura E. Packer; Chao Li; Kojo Abdul-Hadi; Petter Veiby
      Pages: 33 - 36
      Abstract: Publication date: 15 November 2017
      Source:Analytical Biochemistry, Volume 537
      Author(s): Ling Xu, Laura E. Packer, Chao Li, Kojo Abdul-Hadi, Petter Veiby
      The current industry practice for antibody-drug conjugate (ADC) bioanalysis includes quantification of total antibody and antibody-conjugated drug. Here, we report a novel 2-in-1 approach for measuring total antibody and protease-cleavable conjugated drug Monomethyl Auristatin E (MMAE) concurrently. This allows for the determination of the DAR (Drug Antibody Ratio) for in vivo samples, with a 3-orders linear range based on total antibody concentration from 0.1 to 100 μg/mL. Our generic, concurrent method has been cross-validated with the previously established methods in an animal study. This novel approach is applicable to all human IgG1 ADCs with papain cleavable conjugated drug in preclinical studies.

      PubDate: 2017-09-05T19:57:42Z
      DOI: 10.1016/j.ab.2017.08.024
      Issue No: Vol. 537 (2017)
  • Methylene blue as a lignin surrogate in manganese peroxidase reaction
    • Authors: Jeffrey D. Goby; Michael H. Penner; Curtis A. Lajoie; Christine J. Kelly
      Pages: 37 - 40
      Abstract: Publication date: 15 November 2017
      Source:Analytical Biochemistry, Volume 537
      Author(s): Jeffrey D. Goby, Michael H. Penner, Curtis A. Lajoie, Christine J. Kelly
      Manganese peroxidase (MnP) is associated with lignin degradation and is thus relevant to lignocellulosic-utilization technologies. Technological applications require reaction mixture optimization. A surrogate substrate can facilitate this if its susceptibility to degradation is easily monitored and mirrors that of lignin. The dye methylene blue (MB) was evaluated in these respects as a surrogate substrate by testing its reactivity in reaction mixtures containing relevant redox mediators (dicarboxylic acids, fatty acids). Relative rates of MB degradation were compared to available literature reports of lignin degradation under similar conditions, and suggest that MB can be a useful lignin surrogate in MnP systems.

      PubDate: 2017-09-05T19:57:42Z
      DOI: 10.1016/j.ab.2017.08.010
      Issue No: Vol. 537 (2017)
  • An improved amperometric creatinine biosensor based on nanoparticles of
           creatininase, creatinase and sarcosine oxidase
    • Authors: Parveen Kumar; Ranjana Jaiwal; C.S. Pundir
      Pages: 41 - 49
      Abstract: Publication date: 15 November 2017
      Source:Analytical Biochemistry, Volume 537
      Author(s): Parveen Kumar, Ranjana Jaiwal, C.S. Pundir
      An improved amperometric biosensor for detection of creatinine was developed based on immobilization of nanoparticles (NPs) of creatininase (CA), creatinase (CI), and sarcosine oxidase (SOx) onto glassy carbon (GC) electrode. Transmission electron microscopy (TEM) and fourier transform infrared spectroscopy (FTIR) were employed for characterization of enzyme nanoparticles (ENPs). The GC electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) at different stages of its amendment. The biosensor showed optimum response within 2s at pH 6.0 in 0.1 M sodium phosphate buffer and 25 °C, when operated at 1.0 V against Ag/AgCl. Biosensor exhibited wider linear range from 0.01 μM to 12 μM with a limit of detection (LOD) of 0.01 μM. The analytical recoveries of added creatinine in sera were 97.97 ± 0.1% for 0.1 mM and 98.76 ± 0.2% for 0.15 mM, within and between batch coefficients of variation (CV) were 2.06% and 3.09% respectively. A good correlation (R2 = 0.99) was observed between sera creatinine values obtained by standard enzymic colorimetric method and the present biosensor. This biosensor measured creatinine level in sera of apparently healthy subjects and persons suffering from renal and muscular dysfunction. The ENPs electrode lost 10% of its initial activity within 240 days of its regular uses, when stored at 4 °C.

      PubDate: 2017-09-05T19:57:42Z
      DOI: 10.1016/j.ab.2017.08.022
      Issue No: Vol. 537 (2017)
  • A novel method of multiple nucleic acid detection: Real-time RT-PCR
           coupled with probe-melting curve analysis
    • Authors: Yang Han; Shao-Yang Hou; Shang-Zhi Ji; Juan Cheng; Meng-Yue Zhang; Li-Juan He; Xiang-Zhong Ye; Yi-Min Li; Yi-Xuan Zhang
      Pages: 50 - 55
      Abstract: Publication date: 15 November 2017
      Source:Analytical Biochemistry, Volume 537
      Author(s): Yang Han, Shao-Yang Hou, Shang-Zhi Ji, Juan Cheng, Meng-Yue Zhang, Li-Juan He, Xiang-Zhong Ye, Yi-Min Li, Yi-Xuan Zhang
      A novel method, real-time reverse transcription PCR (real-time RT-PCR) coupled with probe-melting curve analysis, has been established to detect two kinds of samples within one fluorescence channel. Besides a conventional TaqMan probe, this method employs another specially designed melting-probe with a 5′ terminus modification which meets the same label with the same fluorescent group. By using an asymmetric PCR method, the melting-probe is able to detect an extra sample in the melting stage effectively while it almost has little influence on the amplification detection. Thus, this method allows the availability of united employment of both amplification stage and melting stage for detecting samples in one reaction. The further demonstration by simultaneous detection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in one channel as a model system is presented in this essay. The sensitivity of detection by real-time RT-PCR coupled with probe-melting analysis was proved to be equal to that detected by conventional real-time RT-PCR. Because real-time RT-PCR coupled with probe-melting analysis can double the detection throughputs within one fluorescence channel, it is expected to be a good solution for the problem of low-throughput in current real-time PCR.

      PubDate: 2017-09-11T12:30:58Z
      DOI: 10.1016/j.ab.2017.08.026
      Issue No: Vol. 537 (2017)
  • A miniaturized assay for kinetic characterization of the Na+-translocating
           NADH:ubiquinone oxidoreductase from Vibrio cholerae
    • Authors: Valentin Muras; Björn Claussen; Hamid Nasiri; Günter Fritz; Julia Steuber
      Pages: 56 - 59
      Abstract: Publication date: 15 November 2017
      Source:Analytical Biochemistry, Volume 537
      Author(s): Valentin Muras, Björn Claussen, Hamid Nasiri, Günter Fritz, Julia Steuber
      We demonstrate the miniaturization of an enzymatic assay for the determination of NADH oxidation and quinone reduction by the Na+ -translocating NADH quinone oxidoreductase (NQR) in the 96-well plate format. The assay is based on the spectrophotometric detection of NADH consumption and quinol formation. We validated the new method with known inhibitors of the NQR and optimized conditions for high-throughput screening as demonstrated by excellent Z-factors well above the accepted threshold (≥0.5). Overall, the method allows the screening and identification of potential inhibitors of the NQR, and rapid characterization of NQR variants obtained by site-specific mutagenesis.

      PubDate: 2017-09-11T12:30:58Z
      DOI: 10.1016/j.ab.2017.08.025
      Issue No: Vol. 537 (2017)
  • Continuous liquid feeding: New method to study pesticides toxicity in
           Drosophila melanogaster
    • Authors: Jefferson J. Soares; Mayara B. Gonçalves; Mateus C. Gayer; Matheus C. Bianchini; Aline C. Caurio; Susana J. Soares; Robson L. Puntel; Rafael Roehrs; Elton L.G. Denardin
      Pages: 60 - 62
      Abstract: Publication date: 15 November 2017
      Source:Analytical Biochemistry, Volume 537
      Author(s): Jefferson J. Soares, Mayara B. Gonçalves, Mateus C. Gayer, Matheus C. Bianchini, Aline C. Caurio, Susana J. Soares, Robson L. Puntel, Rafael Roehrs, Elton L.G. Denardin
      Fly fruit Drosophila melanogaster (DM) has been extensively employed as an in vivo model system to study pesticides toxicity. Pesticide administration to the fly traditionally involves feeding in an agar-gelled feed fly's medium (AM). However, AM method has several limitations such as uncertainty regarding the bioavailability and amount of pesticides ingested. And also high manipulation of the treated flies. We developed a new method of exposure the flies to pesticides, called Continuous Liquid Feeding (CLF). This method successfully delivers food to the flies at much higher concentrations than the AM method, and requires little manipulation of flies under treatment.
      Graphical abstract image

      PubDate: 2017-09-11T12:30:58Z
      DOI: 10.1016/j.ab.2017.08.016
      Issue No: Vol. 537 (2017)
  • Chemiluminescence immunoassays for estradiol and ethinylestradiol based on
           new biotinylated estrogen derivatives
    • Authors: Hussein Kanso; Nicolas Inguimbert; Georges Istamboulie; Lise Barthelmebs; Carole Calas-Blanchard; Thierry Noguer
      Pages: 63 - 68
      Abstract: Publication date: 15 November 2017
      Source:Analytical Biochemistry, Volume 537
      Author(s): Hussein Kanso, Nicolas Inguimbert, Georges Istamboulie, Lise Barthelmebs, Carole Calas-Blanchard, Thierry Noguer
      New chemiluminescence-based immunoassays for sensitive detection of 17-β estradiol (E2) and ethinylestradiol (EE2) are described on the basis of the use of biotinylated estrogen derivatives. Estrogen derivatives bearing a carboxylic group (E2-COOH and EE2-COOH) on C-3 position were synthesized, covalently bound to aminated biotin and subsequently immobilized on avidin-coated microtiter plates. The assay principle was based on competition between free and immobilized estrogens for their binding to primary antibodies, with subsequent revelation using horseradish peroxidase (HRP)-labeled secondary antibodies. Under optimized conditions, the chemiluminescence immunoassays showed a highly sensitive response to E2 and EE2, with respective detection limits of 0.5 and 1.2 ng L−1. The LOD achieved using biotinylated E2 was in the same order of magnitude as those obtained using commercially available E2-bovine serum albumin conjugate (E2-BSA). The developed devices were successfully applied to analysis wastewater treatment plants effluents (WWTP) with negligible matrix effect.

      PubDate: 2017-09-11T12:30:58Z
      DOI: 10.1016/j.ab.2017.08.023
      Issue No: Vol. 537 (2017)
  • Adsorption effects of the doping relevant peptides Insulin Lispro,
           Synachten, TB-500 and GHRP 5
    • Authors: Péter Judák; Peter Van Eenoo; Koen Deventer
      Pages: 69 - 71
      Abstract: Publication date: 15 November 2017
      Source:Analytical Biochemistry, Volume 537
      Author(s): Péter Judák, Peter Van Eenoo, Koen Deventer
      The tendency of peptides to adsorb to surfaces can raise a concern in variety of analytical fields where the qualitative/quantitative measurement of low concentration analytes (ng/mL-pg/mL) is required. To demonstrate the importance of using the optimal glassware/plasticware, four doping relevant model peptides (GHRP 5, TB-500, Insulin Lispro, Synachten) were chosen and their recovery from various surfaces were evaluated. Our experiments showed that choosing expensive consumables with low-bind characteristics is not beneficial in all cases. A careful selection of the consumables based on the evaluation of the physico/chemical features of the peptide is recommended.
      Graphical abstract image

      PubDate: 2017-09-11T12:30:58Z
      DOI: 10.1016/j.ab.2017.09.003
      Issue No: Vol. 537 (2017)
  • IEF peptide fractionation method combined to shotgun proteomics enhances
           the exploration of rice milk proteome
    • Authors: Marcello Manfredi; Jessica Brandi; Eleonora Conte; Paolo Pidutti; Fabio Gosetti; Elisa Robotti; Emilio Marengo; Daniela Cecconi
      Pages: 72 - 77
      Abstract: Publication date: 15 November 2017
      Source:Analytical Biochemistry, Volume 537
      Author(s): Marcello Manfredi, Jessica Brandi, Eleonora Conte, Paolo Pidutti, Fabio Gosetti, Elisa Robotti, Emilio Marengo, Daniela Cecconi
      We conducted a proteomics study in order to detect the proteomic method which provides the most complete characterization of the proteins of rice milk. In particular, we compared the results obtained from LC-MS/MS after protein precipitation with acetone or TCA, as well as the results obtained from LC-MS/MS after protein prefractionation based on SDS-PAGE (GeLC-MS/MS) or ProteoMiner™ technology (ProteoMiner-LC-MS/MS), and after peptide prefractionation based on IEF (pIEF-LC-MS/MS). A total of 158 protein species have been detect in rice milk. The physical-chemical analysis and classification of the identified proteins were also reported. In particular, we showed that pIEF-LC-MS/MS method led to a significant increase in the proteome coverage, allowing the identification of a total of 96 proteins of milk rice. This study demonstrates the utility of a prefractionation step based on pIEF before the shotgun proteomic analysis and offers an in-depth insight into the rice milk proteome.

      PubDate: 2017-09-11T12:30:58Z
      DOI: 10.1016/j.ab.2017.08.021
      Issue No: Vol. 537 (2017)
  • Genotyping of single nucleotide polymorphism by probe-gated silica
    • Authors: Meltem Ercan; Veli C. Ozalp; Bilge G. Tuna
      Pages: 78 - 83
      Abstract: Publication date: 15 November 2017
      Source:Analytical Biochemistry, Volume 537
      Author(s): Meltem Ercan, Veli C. Ozalp, Bilge G. Tuna
      The development of simple, reliable, and rapid approaches for molecular detection of common mutations is important for prevention and early diagnosis of genetic diseases, including Thalessemia. Oligonucleotide-gated mesoporous nanoparticles-based analysis is a new platform for mutation detection that has the advantages of sensitivity, rapidity, accuracy, and convenience. A specific mutation in β-thalassemia, one of the most prevalent inherited diseases in several countries, was used as model disease in this study. An assay for detection of IVS110 point mutation (A > G reversion) was developed by designing probe-gated mesoporous silica nanoparticles (MCM-41) loaded with reporter fluorescein molecules. The silica nanoparticles were characterized by AFM, TEM and BET analysis for having 180 nm diameter and 2.83 nm pore size regular hexagonal shape. Amine group functionalized nanoparticles were analysed with FTIR technique. Mutated and normal sequence probe oligonucleotides)about 12.7 nmol per mg nanoparticles) were used to entrap reporter fluorescein molecules inside the pores and hybridization with single stranded DNA targets amplified by PCR gave different fluorescent signals for mutated targets. Samples from IVS110 mutated and normal patients resulted in statistically significant differences when the assay procedure were applied.

      PubDate: 2017-09-18T00:37:50Z
      DOI: 10.1016/j.ab.2017.09.004
      Issue No: Vol. 537 (2017)
  • A multiplex ARMS PCR approach to detection of common β-globin gene
    • Authors: Kanchan K. Mishra; Parizad Patel; Dipal S. Bhukhanvala; Avani Shah; Kanjaksha Ghosh
      Pages: 93 - 98
      Abstract: Publication date: 15 November 2017
      Source:Analytical Biochemistry, Volume 537
      Author(s): Kanchan K. Mishra, Parizad Patel, Dipal S. Bhukhanvala, Avani Shah, Kanjaksha Ghosh
      Background β-thalassaemia is a group of inherited single-gene disorders worldwide. Each ethnic population has its own common mutations. Heterogeneity of β-thalassaemia mutations in multi-ethnic population of Surat, makes molecular diagnosis expensive and time consuming. Methods Specific primers were used to differentiate four common mutations, IVS I-5 (G→C), Codon 41/42 (- TCTT), 619-bp deletion and FS 8/9 (+G), by a simple PCR involving a multiplex amplification refractory mutation system. Results Several high prevalence β-Thalassemia trait groups constituted by Muslims, Patels, Sindhis, ModhBanias, and Mahayavanshi. Four most common mutations detected in them are IVS I-5 (G→C), Codon 41/42 (- TCTT), 619-bp deletion and FS 8/9 (+G). We identified each of these β-thalassemia mutations in multiplexed ARMS from positive control samples. Our multiplex-ARMS-PCR system was first standardized on positive DNA samples with above known four most common β-thalassemia mutations, and these positive samples had been diagnosed with β-thalassemia and also all these samples belonged to Surat ethnic groups. The system was subsequently tested on 110 blood samples from different ethnic backgrounds with unknown β-thalassemia mutations which were in all specimens. Conclusion The ARMS multiplex system was found reliable, cost effective, fast and most applicable for mutation screening of Thalassemia in Surat populations.

      PubDate: 2017-09-18T00:37:50Z
      DOI: 10.1016/j.ab.2017.06.014
      Issue No: Vol. 537 (2017)
  • A multiplex protein-free lateral flow assay for detection of microRNAs
           based on unmodified molecular beacons
    • Authors: Atefeh Javani; Fatemeh Javadi-Zarnaghi; Mohammad Javad Rasaee
      Pages: 99 - 105
      Abstract: Publication date: 15 November 2017
      Source:Analytical Biochemistry, Volume 537
      Author(s): Atefeh Javani, Fatemeh Javadi-Zarnaghi, Mohammad Javad Rasaee
      Lateral flow assays (LFAs) have promising potentials for point-of-care applications. Recently, many LFAs have been reported that are based on hybridization of oligonucleotide strands. Mostly, biotinylated capture DNAs are immobilized on the surface of a nitrocellulose membrane via streptavidin interactions. During the assay, stable colorful complexes get formed that are visible by naked eyes. Here, we present an inexpensive and unique design of LFA that applies unmodified oligonucleotides at capture lines. The presented LFA do not utilize streptavidin or any other affinity protein. We employ structural switch of molecular beacons (MB) in combination with base stacking hybridization (BSH) phenomenon. The unique design of the reported LFA provided high selectivity for target oligonucleotides. We validated potential applications of the system for detection of DNA mimics of two microRNAs in multiplex assays.
      Graphical abstract image

      PubDate: 2017-09-18T00:37:50Z
      DOI: 10.1016/j.ab.2017.09.005
      Issue No: Vol. 537 (2017)
  • Isolation of mouse chromaffin secretory vesicles and their division into
           12 fractions
    • Authors: Marta R. Pardo; Judith Estévez-Herrera; Leandro Castañeyra; Ricardo Borges; José David Machado
      Pages: 1 - 7
      Abstract: Publication date: 1 November 2017
      Source:Analytical Biochemistry, Volume 536
      Author(s): Marta R. Pardo, Judith Estévez-Herrera, Leandro Castañeyra, Ricardo Borges, José David Machado
      The study of chromaffin secretory vesicles (SVs) has contributed immensely to our understanding of exocytosis. These organelles, also called chromaffin granules, are a specific type of large dense secretory vesicle found in many endocrine cells and neurons. Traditionally, they have been isolated from bovine adrenal glands due to the large number of SVs that can be obtained from this tissue. However, technical advances now make it possible to obtain very pure preparations of SVs from mice, which is particular interesting for functional studies given the availability of different genetically modified strains of mice. Despite the small size of the mouse adrenal medulla (400–500 μm and less than 2 mg in weight), we have successfully carried out functional studies on SVs isolated from WT and knockout mice. As such, we present here our method to purify crude vesicles and to fractionate mouse chromaffin SVs, along with examples of their functional characterization.

      PubDate: 2017-08-15T19:30:59Z
      DOI: 10.1016/j.ab.2017.07.026
      Issue No: Vol. 536 (2017)
  • A method for extracting and characterizing RNA from urine: For downstream
           PCR and RNAseq analysis
    • Authors: Kun Zhou; Monique A. Spillman; Kian Behbakht; Julia M. Komatsu; Juan E. Abrahante; Douglas Hicks; Brent Schotl; Evan Odean; Kenneth L. Jones; Michael W. Graner; Lynne T. Bemis
      Pages: 8 - 15
      Abstract: Publication date: 1 November 2017
      Source:Analytical Biochemistry, Volume 536
      Author(s): Kun Zhou, Monique A. Spillman, Kian Behbakht, Julia M. Komatsu, Juan E. Abrahante, Douglas Hicks, Brent Schotl, Evan Odean, Kenneth L. Jones, Michael W. Graner, Lynne T. Bemis
      Readily accessible samples such as urine or blood are seemingly ideal for differentiating and stratifying patients; however, it has proven a daunting task to identify reliable biomarkers in such samples. Noncoding RNA holds great promise as a source of biomarkers distinguishing physiologic wellbeing or illness. Current methods to isolate and characterize RNA molecules in urine are limited. In this proof of concept study, we present a method to extract and identify small noncoding RNAs in urine. Initially, quantitative reverse transcription PCR was applied to confirm the presence of microRNAs in total RNA extracted from urine. Once the presence of micro RNA in urine was confirmed, we developed a method to scale up RNA extraction to provide adequate amounts of RNA for next generation sequence analysis. The method described in this study is applicable to detecting a broad range of small noncoding RNAs in urine; thus, they have wide applicability for health and disease analyses.

      PubDate: 2017-08-26T19:46:53Z
      DOI: 10.1016/j.ab.2017.08.003
      Issue No: Vol. 536 (2017)
  • Real-time qRT-PCR assay for the detection of miRNAs using bi-directional
           extension sequences
    • Authors: Kyung Jin Kim; Jiwon Kwak; Jae-Hoon Lee; Soo Suk Lee
      Pages: 32 - 35
      Abstract: Publication date: 1 November 2017
      Source:Analytical Biochemistry, Volume 536
      Author(s): Kyung Jin Kim, Jiwon Kwak, Jae-Hoon Lee, Soo Suk Lee
      Highly specific detection of miRNAs was performed using a novel bi-directional extension (BDE) assay. After reverse transcription, the cDNA was hybridized to a uniquely designed specific BDE sequence; this cDNA-BDE hybrid forms the PCR template. The PCR template was amplified in a SYBR Green-based quantitative real-time PCR. The miR-145 in human brain total RNA could be detected quantitatively in the range of seven orders of magnitude with high linearity and reproducibility. This innovative BDE assay has several performance advantages over the poly(A) tailing method that include lower CT values, clear gel electrophoresis images, and distinct nucleotide peaks in sequencing chromatograms.

      PubDate: 2017-08-26T19:46:53Z
      DOI: 10.1016/j.ab.2017.08.006
      Issue No: Vol. 536 (2017)
  • DNA aptamer identification and characterization for E. coli O157
           detection using cell based SELEX method
    • Authors: Masoum Amraee; Mana Oloomi; Afsaneh Yavari; Saeid Bouzari
      Pages: 36 - 44
      Abstract: Publication date: 1 November 2017
      Source:Analytical Biochemistry, Volume 536
      Author(s): Masoum Amraee, Mana Oloomi, Afsaneh Yavari, Saeid Bouzari
      Escherichia coli (E. coli) O157:H7 is a foodborne pathogen that causes symptoms in humans. Its rapid identification should be considered to avoid toxic effects of the pathogen. In this study, systematic evolution of ligands by exponential enrichment using whole cells (Cell-SELEX) method was used for recognizing E. coli strain, O157 by single-stranded DNA library of aptamer. Nine rounds of cell-selex procedure were applied using O157, as a whole-cell target, with O42, K12, Top10, DH5α E. coli cells, Shigella flexneri and Salmonella typhi as counterparts. The specific interaction between selected DNA aptamers and targeted cell was assessed. After applying six rounds of SELEX for selection of DNA aptamers, the candidate sequences were obtained. Finally, specific aptamer was selected as an ideal aptamer for detection and capturing of E. coli O157. Dissociation constant of the selected aptamer were calculated (107.6 ± 67.8 pM). In addition, the secondary structure prediction and cross reactivity assays were performed. The isolated aptamer efficiency was confirmed and it was shown that the new DNA aptamer sequence has the ability to use for detection. This specific O157:H7 aptamer have the potential for application as a diagnostic ligand and could be used for detection of the related food borne diseases.

      PubDate: 2017-08-26T19:46:53Z
      DOI: 10.1016/j.ab.2017.08.005
      Issue No: Vol. 536 (2017)
  • Enhanced post wash retention of combed DNA molecules by varying multiple
           combing parameters
    • Authors: Hemendra Yadav
      Pages: 45 - 50
      Abstract: Publication date: 1 November 2017
      Source:Analytical Biochemistry, Volume 536
      Author(s): Hemendra Yadav
      Recent advances in genomics have created a need for efficient techniques for deciphering information hidden in various genomes. Single molecule analysis is one such technique to understand molecular processes at single molecule level. Fiber- FISH performed with the help of DNA combing can help us in understanding genetic rearrangements and changes in genome at single DNA molecule level. For performing Fiber-FISH we need high retention of combed DNA molecules post wash as Fiber-FISH requires profuse washing. We optimized combing process involving combing solution, method of DNA mounting on glass slides and coating of glass slides to enhance post-wash retention of DNA molecules. It was found that average number of DNA molecules observed post-wash per field of view was maximum with our optimized combing solution. APTES coated glass slides showed lesser retention than PEI surface but fluorescent intensity was higher in case of APTES coated surface. Capillary method used to mount DNA on glass slides also showed lesser retention but straight DNA molecules were observed as compared to force flow method.

      PubDate: 2017-08-26T19:46:53Z
      DOI: 10.1016/j.ab.2017.08.008
      Issue No: Vol. 536 (2017)
  • Graphene oxide layer decorated gold nanoparticles based immunosensor for
           the detection of prostate cancer risk factor
    • Authors: Mintu Pal; Raju Khan
      Pages: 51 - 58
      Abstract: Publication date: 1 November 2017
      Source:Analytical Biochemistry, Volume 536
      Author(s): Mintu Pal, Raju Khan
      In this work, we report a novel electrochemical immunosensor based on gold nanoparticles deposited on the surface of graphene oxide layers and was used for immobilization of monoclonal anti-PSA antibody via EDC/NHS coupling method to detect prostate-specific antigen (PSA), a valuable biomarker for early detection of prostate cancer. To confirm the functionality of the antibody, we performed immunofluorescence staining using human prostate adenocarcinoma cells, LNCaP. Scanning Electron Microscopy (SEM), cyclic voltammetry, and other electrochemical techniques were used to characterize the resulting electrode surface. Unlike the previous research, this novel immunosensor functions very well with a low detection limit of 0.24 fgmL−1 at signal to noise ratio of 3; furthermore, it exhibits a significantly increased electron transfer and high sensitivity of 5.4 μA fgmL−1 with regression coefficient (R2 = 0.99) toward PSA. The immunosensor was verified for selective and accurate detection of PSA in human serum with recovery of 97.67%. Overall, data suggested that our developed biosensor holds great promise as a useful alternative diagnostic tool for the detection of different cancer biomarkers, in particular PSA present in biological samples.

      PubDate: 2017-08-26T19:46:53Z
      DOI: 10.1016/j.ab.2017.08.001
      Issue No: Vol. 536 (2017)
  • A miniaturized peptidyl-prolyl isomerase enzyme assay
    • Authors: Mirella Vivoli; Julien Renou; Arnaud Chevalier; Isobel H. Norville; Suraya Diaz; Christina Juli; Helen Atkins; Ulrike Holzgrabe; Pierre-Yves Renard; Mitali Sarkar-Tyson; Nicholas J. Harmer
      Pages: 59 - 68
      Abstract: Publication date: 1 November 2017
      Source:Analytical Biochemistry, Volume 536
      Author(s): Mirella Vivoli, Julien Renou, Arnaud Chevalier, Isobel H. Norville, Suraya Diaz, Christina Juli, Helen Atkins, Ulrike Holzgrabe, Pierre-Yves Renard, Mitali Sarkar-Tyson, Nicholas J. Harmer
      Prolyl-peptidyl isomerases (PPIases) are enzymes that are found in all living organisms. They form an essential part of the cellular protein folding homeostasis machinery. PPIases are associated with many important human diseases, e.g. cardiovascular disease, cancer and Alzheimer's. The development of novel PPIase inhibitors has been limited by the lack of a rapid, laboratory-based assay for these enzymes, as their substrates and products are challenging to distinguish. A well described continuous assay, coupled with the hydrolysis of a peptide by chymotrypsin is highly effective, but comparatively slow. To address this, we developed an improved version of the traditional assay using a temperature controlled plate reader. This assay allows semi-automated medium throughput assays in an academic laboratory for 84 samples per day. The assay shows lower errors, with an average Z′ of 0.72. We further developed the assay using a fluorogenic peptide-based FRET probe. This provides an extremely sensitive PPIase assay using substrate at 200 nM, which approaches single turnover conditions. The fluorescent probe achieves an excellent quenching efficiency of 98.6%, and initial experiments showed acceptable Z′ of 0.31 and 0.30 for cyclophilin A and hFKBP12 respectively. The assays provide an improved toolset for the quantitative, biochemical analysis of PPIases.

      PubDate: 2017-09-23T23:34:00Z
      DOI: 10.1016/j.ab.2017.08.004
      Issue No: Vol. 536 (2017)
  • Expression and purification of functional insulin and insulin-like growth
           factor 1 holoreceptors from mammalian cells
    • Authors: Richard J. Delle Bovi; W. Todd Miller
      Pages: 69 - 77
      Abstract: Publication date: 1 November 2017
      Source:Analytical Biochemistry, Volume 536
      Author(s): Richard J. Delle Bovi, W. Todd Miller
      The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) are receptor tyrosine kinases (RTKs) involved in the regulation of many important cellular processes. The current proposed models of activation are derived from structural studies using soluble extracellular domains and cytoplasmic tyrosine kinase domains. Preparations of full length IR and IGF1R have been hampered by the need for unconventional affinity chromatography resins and/or harsh eluting conditions. Here, we present a purification protocol to obtain full-length, detergent solubilized IR and IGF1R at quantities suitable for biochemical and structural characterization. We screened a panel of 24 structurally diverse detergents for optimal ligand activation. The receptors purified in n-dodecyl-β-D-maltoside showed ligand-stimulated autophosphorylation and kinase activity, suggesting an intact transmembrane signaling mechanism. This convenient purification protocol can be used to produce high quantities of IR, IGF1R, or other RTKs, and can be adapted for other challenging membrane proteins.

      PubDate: 2017-09-23T23:34:00Z
      DOI: 10.1016/j.ab.2017.08.011
      Issue No: Vol. 536 (2017)
  • New method for estimating clustering of DNA lesions induced by
           physical/chemical mutagens using fluorescence anisotropy
    • Authors: Ken Akamatsu; Naoya Shikazono; Takeshi Saito
      Pages: 78 - 89
      Abstract: Publication date: 1 November 2017
      Source:Analytical Biochemistry, Volume 536
      Author(s): Ken Akamatsu, Naoya Shikazono, Takeshi Saito
      We have developed a new method for estimating the localization of DNA damage such as apurinic/apyrimidinic sites (APs) on DNA using fluorescence anisotropy. This method is aimed at characterizing clustered DNA damage produced by DNA-damaging agents such as ionizing radiation and genotoxic chemicals. A fluorescent probe with an aminooxy group (AlexaFluor488) was used to label APs. We prepared a pUC19 plasmid with APs by heating under acidic conditions as a model for damaged DNA, and subsequently labeled the APs. We found that the observed fluorescence anisotropy (r obs) decreases as averaged AP density (λ AP: number of APs per base pair) increases due to homo-FRET, and that the APs were randomly distributed. We applied this method to three DNA-damaging agents, 60Co γ-rays, methyl methanesulfonate (MMS), and neocarzinostatin (NCS). We found that r obs–λ AP relationships differed significantly between MMS and NCS. At low AP density (λ AP < 0.001), the APs induced by MMS seemed to not be closely distributed, whereas those induced by NCS were remarkably clustered. In contrast, the AP clustering induced by 60Co γ-rays was similar to, but potentially more likely to occur than, random distribution. This simple method can be used to estimate mutagenicity of ionizing radiation and genotoxic chemicals.

      PubDate: 2017-09-23T23:34:00Z
      DOI: 10.1016/j.ab.2017.08.007
      Issue No: Vol. 536 (2017)
  • The role of human monoacylglycerol lipase (hMAGL) binding pocket in
           breakup of unsaturated phospholipid membranes
    • Authors: Ioannis Karageorgos; Vitalii I. Silin; Nikolai Zvonok; John Marino; David R. Janero; Alexandros Makriyannis
      Pages: 90 - 95
      Abstract: Publication date: 1 November 2017
      Source:Analytical Biochemistry, Volume 536
      Author(s): Ioannis Karageorgos, Vitalii I. Silin, Nikolai Zvonok, John Marino, David R. Janero, Alexandros Makriyannis
      Human monoacylglycerol lipase (hMAGL) plays a key role in homeostatic tuning of the endocannabinoid signaling system and supports aggressive tumorogenesis, making this enzyme a promising therapeutic target. hMAGL features a membrane-associated lid domain that regulates entry of endocannabinoid lipid substrates into the hydrophobic channel accessing the active site, likely from the membrane bilayer. The present work applied simultaneous surface plasmon resonance and electrochemical impedance spectroscopy measurements to show that, in absence of the substrate, hMAGL can remove phospholipid molecules from the membrane and, thereby, disintegrate pre-formed, intact, tethered phospholipid bilayer membrane mimetics (tBLMs) composed of unsaturated phosphatidylcholines. To probe the mechanism of hMAGL-induced on tBLMs compromise, we investigated the effect of wild type and mutant hMAGLs and hMAGL rendered catalytically inactive, as a function of concentration and in the presence of chemically distinct active-site inhibitors. Our data show that hMAGL's lid domain and hydrophobic substrate-binding pocket play important roles in hMAGL-induced bilayer lipid mobilization, whereas hydrolytic activity of the enzyme does not appear to be a factor.

      PubDate: 2017-09-23T23:34:00Z
      DOI: 10.1016/j.ab.2017.08.009
      Issue No: Vol. 536 (2017)
  • NADPH oxidase activity: Spectrophotometric determination of superoxide
           using pyrogallol red
    • Authors: J. Cortés-Ríos; M.J. Torres; M.P. Campos-Bustamante; J. Romero-Parra; M.E. Letelier; D. Pessoa-Mahana; H. Chung; M. Faúndez
      Pages: 96 - 100
      Abstract: Publication date: 1 November 2017
      Source:Analytical Biochemistry, Volume 536
      Author(s): J. Cortés-Ríos, M.J. Torres, M.P. Campos-Bustamante, J. Romero-Parra, M.E. Letelier, D. Pessoa-Mahana, H. Chung, M. Faúndez
      A simple and fast spectrophotometric methodology able to quantify superoxide released by NADPH oxidase from differentiated promyelocytic leukaemia (HL-60) cells using pyrogallol red is described.The latter is based on the known stoichiometry of the reaction between superoxide and pyrogallol red and the inability of pyrogallol red to react with hydrogen peroxide. In addition, we developed a 96-wells microplate-based method able to determine NADPH oxidase activity. Using this method, we determined pharmacological properties of the NADPH oxidase inhibitors VAS2870 and diphenyleneiodonium and the obtained IC50 values were in good agreement with previous reported data. NOX2 is highly expressed in differentiated promyelocytic leukaemia cells, whereas other isoforms are not detected or expressed at low amounts. Likewise, this methodology may be a useful assay for NOX2 inhibitor screening. NADPH oxidases are involved in several physiological and pathological processes, rendering its pharmacological modulation an attractive research target. In this context, this simple assay can be used for NADPH oxidase inhibitor screening as well as aiding in the study of different biological conditions that involve NADPH oxidase activity.
      Graphical abstract image

      PubDate: 2017-09-23T23:34:00Z
      DOI: 10.1016/j.ab.2017.08.013
      Issue No: Vol. 536 (2017)
  • Biosensors based on β-galactosidase enzyme: Recent advances and
    • Authors: Shiv K. Sharma; Roger M. Leblanc
      Pages: 1 - 11
      Abstract: Publication date: 15 October 2017
      Source:Analytical Biochemistry, Volume 535
      Author(s): Shiv K. Sharma, Roger M. Leblanc
      Many industries are striving for the development of more reliable and robust β-galactosidase biosensors that exhibit high response rate, increased detection limit and enriched useful lifetime. In a newfangled technological atmosphere, a trivial advantage or disadvantage of the developed biosensor may escort to the survival and extinction of the industry. Several alternative strategies to immobilize β-galactosidase enzyme for their utilization in biosensors have been developed in recent years in the quest of maximum utility by controlling the defects seen in the previous biosensors. The overwhelming call for on-line measurement of different sample constituents has directed science and industry to search for best practical solutions and biosensors are witnessed as the best prospect. The main objective of this paper is to serve as a narrow footbridge by comparing the literary works on the β-galactosidase biosensors, critically analyze their use in the construction of best biosensor by showing the pros and cons of the predicted methods for the practical use of biosensors.

      PubDate: 2017-07-27T13:25:27Z
      DOI: 10.1016/j.ab.2017.07.019
      Issue No: Vol. 535 (2017)
  • An enzyme-linked immunosorbent assay for the detection of diacetyl
    • Authors: Lucia Marri; Anita M. Jansson; Caspar E. Christensen; Ole Hindsgaul
      Pages: 12 - 18
      Abstract: Publication date: 15 October 2017
      Source:Analytical Biochemistry, Volume 535
      Author(s): Lucia Marri, Anita M. Jansson, Caspar E. Christensen, Ole Hindsgaul
      Diacetyl (2,3-butanedione) is an important metabolic marker of several cancers, as well as an important off-flavour component produced during fermentation. As a small molecule in a complex mixture with many other analytes, existing methods for identification and quantitation of diacetyl invariably involves a chromatographic separation step followed by signal integration with an appropriate stoichiometric detector. Here we demonstrate that the chemical reaction of diacetyl with a 1,2-phenylenediamine derivative yields a chemical adduct, 1,4-quinoxaline which can be conjugated on BSA. The BSA-diacetyl adduct can be used to select an adduct-specific monoclonal antibody in a Fab-format from a 45-billion member phage-display library. The availability of this antibody allowed the development of an enzyme-linked immunosorbent assay for diacetyl, based on the 1,4-quinoxaline competition for the antibodies with the diacetyl adduct immobilized on the plate. The described ELISA assay can detect the captured diacetyl in micromolar concentrations, both in water samples and in cell culture medium.

      PubDate: 2017-07-27T13:25:27Z
      DOI: 10.1016/j.ab.2017.07.021
      Issue No: Vol. 535 (2017)
  • Colorimetric sensing of selenocystine using gold nanoparticles
    • Authors: Liyang Liu; Xia Wang; Juan Yang; Yan Bai
      Pages: 19 - 24
      Abstract: Publication date: 15 October 2017
      Source:Analytical Biochemistry, Volume 535
      Author(s): Liyang Liu, Xia Wang, Juan Yang, Yan Bai
      We present a highly selective and sensitive colorimetric method for the detection of selenocystine (SeCys) coexisting with other amino acids, especially cysteine (Cys) using the gold nanoparticles (AuNPs). Firstly, Cys was oxidized to cystine (Cys-Cys) by dissolved oxygen under Cu2+ catalysis in the pre-reaction, which eliminated the interference of Cys in the SeCys sensing process. Then SeCys induced the rapid aggregation of AuNPs through Au-Se bond and complex formation of Cu2+-SeCys in the colorimetric reaction, in which the color change of AuNPs from red to blue or purple with the naked eye detection or with a UV-vis spectrophotometric determination. The concentration of SeCys was quantified by the value at 670 nm from the second-derivative SPR absorbance spectrum. The linear range was from 2 μM to 14 μM with correlation coefficient of 0.999 and a detection limit (LOD) was 0.14 μM. Moreover, the colorimetric response of AuNPs exhibited remarkable specificity to SeCys coexisting with 18 amino acids in a simulation sample, in which the total concentration of Cys and Cys-Cys was less than 15 μM and the each concentration of other 16 common amino acids was 10 μM.
      Graphical abstract image

      PubDate: 2017-07-27T13:25:27Z
      DOI: 10.1016/j.ab.2017.07.020
      Issue No: Vol. 535 (2017)
  • Beta-hairpin hydrogels as scaffolds for high-throughput drug discovery in
           three-dimensional cell culture
    • Authors: Peter Worthington; Katherine M. Drake; Zhiqin Li; Andrew D. Napper; Darrin J. Pochan; Sigrid A. Langhans
      Pages: 25 - 34
      Abstract: Publication date: 15 October 2017
      Source:Analytical Biochemistry, Volume 535
      Author(s): Peter Worthington, Katherine M. Drake, Zhiqin Li, Andrew D. Napper, Darrin J. Pochan, Sigrid A. Langhans
      Automated cell-based high-throughput screening (HTS) is a powerful tool in drug discovery, and it is increasingly being recognized that three-dimensional (3D) models, which more closely mimic in vivo-like conditions, are desirable screening platforms. One limitation hampering the development of 3D HTS is the lack of suitable 3D culture scaffolds that can readily be incorporated into existing HTS infrastructure. We now show that β-hairpin peptide hydrogels can serve as a 3D cell culture platform that is compatible with HTS. MAX8 β-hairpin peptides can physically assemble into a hydrogel with defined porosity, permeability and mechanical stability with encapsulated cells. Most importantly, the hydrogels can then be injected under shear-flow and immediately reheal into a hydrogel with the same properties exhibited prior to injection. The post-injection hydrogels are cell culture compatible at physiological conditions. Using standard HTS equipment and medulloblastoma pediatric brain tumor cells as a model system, we show that automatic distribution of cell-peptide mixtures into 384-well assay plates results in evenly dispensed, viable MAX8-cell constructs suitable for commercially available cell viability assays. Since MAX8 peptides can be functionalized to mimic the microenvironment of cells from a variety of origins, MAX8 peptide gels should have broad applicability for 3D HTS drug discovery.

      PubDate: 2017-08-05T02:45:08Z
      DOI: 10.1016/j.ab.2017.07.024
      Issue No: Vol. 535 (2017)
  • ESCORTing proteins directly from whole cell-lysate for single-molecule
    • Authors: Shwetha Srinivasan; Jagadish P. Hazra; Gayathri S. Singaraju; Debadutta Deb; Sabyasachi Rakshit
      Pages: 35 - 42
      Abstract: Publication date: 15 October 2017
      Source:Analytical Biochemistry, Volume 535
      Author(s): Shwetha Srinivasan, Jagadish P. Hazra, Gayathri S. Singaraju, Debadutta Deb, Sabyasachi Rakshit
      We have developed a method for Enzymatic Sortase-assisted Covalent Orientation-specific Restraint Tethering (ESCORT) recombinant proteins onto surfaces directly from cell-lysate. With an improved surface passivation method, we obviate the cumbersome purification steps even for single molecule studies that demand high purity in the sample. We demonstrated high-specificity of the method, high-passivity of the surface and uncompromised functional integrity of anchored proteins using single molecule fluorescence and force-mapping. We anticipate that this method will substantially reduce the investment by way of time, money and energy in the area of single molecule studies.

      PubDate: 2017-08-05T02:45:08Z
      DOI: 10.1016/j.ab.2017.07.022
      Issue No: Vol. 535 (2017)
  • Plasmid DNA purification by zirconia magnetic nanocomposite
    • Authors: Mohammad Saraji; Shila Yousefi Majid Talebi
      Abstract: Publication date: Available online 7 October 2017
      Source:Analytical Biochemistry
      Author(s): Mohammad Saraji, Shila Yousefi, Majid Talebi
      A zirconia magnetic nanocomposite was prepared by a co-precipitation method in one step. The synthesized nanocomposite was characterized by scanning electron microscopy, energy-dispersive X-ray and Fourier transform infrared spectroscopy. The nanocomposite was applied for the adsorption/desorption of tobacco plant DNA (as a model). Experimental parameters controlling adsorption efficiency, including pH of binding solution, extraction time, the amount of nanocomposite and ionic strength, were optimized. To obtain high desorption efficiency, the effects of pH, the ionic strength of elution buffer and desorption time were investigated. The nanocomposite provided the adsorption capacity of 53.5 mg g−1 for DNA. The adsorption and desorption efficiency of the sorbent was found to be greater than 98 and 81%, respectively. The zirconia magnetic nanocomposite was used for the purification of plasmid DNA (pDNA) from Escherichia coli cell culture. The yield and purity of pDNA obtained by the method were compared to those obtained by the phenol-chloroform solvent extraction and a commercial kit.

      PubDate: 2017-10-08T07:46:38Z
  • Application of RT-PCR and MALDI-TOF MS for the detection of RNA luteovirus
    • Authors: Hideyuki Kajiwara; Ritsuko Murakami
      Abstract: Publication date: Available online 6 October 2017
      Source:Analytical Biochemistry
      Author(s): Hideyuki Kajiwara, Ritsuko Murakami
      There is a need for rapid and less expensive methods to identify RNA viruses, including luteoviruses, for practical use in agriculture and quarantine. The mass spectrometric cleaved amplified polymorphic sequence (MS-CAPS) method, which detects enzymatically cleaved amplicons by matrix-assisted laser desorption/ionization mass spectrometry, was herein used together with a short RT-PCR to detect luteovirus in only 90 min. In addition, the matrixes 2′,4′,6′-trihydroxyacetophene and 3-hydroxypicolinic acid were compared for their effectiveness in the analysis of short single-stranded biotinylated DNA obtained by a MS-CAPS reaction.

      PubDate: 2017-10-08T07:46:38Z
School of Mathematical and Computer Sciences
Heriot-Watt University
Edinburgh, EH14 4AS, UK
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Fax: +00 44 (0)131 4513327
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