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Showing 1 - 200 of 3080 Journals sorted alphabetically
A Practical Logic of Cognitive Systems     Full-text available via subscription   (Followers: 7)
AASRI Procedia     Open Access   (Followers: 15)
Academic Pediatrics     Hybrid Journal   (Followers: 25, SJR: 1.402, h-index: 51)
Academic Radiology     Hybrid Journal   (Followers: 22, SJR: 1.008, h-index: 75)
Accident Analysis & Prevention     Partially Free   (Followers: 86, 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: 363, 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: 229, 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: 1)
Acta Poética     Open Access   (Followers: 4)
Acta Psychologica     Hybrid Journal   (Followers: 24, 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)
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Actas Urológicas Españolas     Full-text available via subscription   (Followers: 4, SJR: 0.383, h-index: 19)
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Actualites Pharmaceutiques     Full-text available via subscription   (Followers: 5, SJR: 0.141, h-index: 3)
Actualites Pharmaceutiques Hospitalieres     Full-text available via subscription   (Followers: 4, SJR: 0.112, h-index: 2)
Acupuncture and Related Therapies     Hybrid Journal   (Followers: 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: 6)
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: 132, 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: 26, 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 Cell Aging and Gerontology     Full-text available via subscription   (Followers: 4)
Advances in Cellular and Molecular Biology of Membranes and Organelles     Full-text available via subscription   (Followers: 12)
Advances in Chemical Engineering     Full-text available via subscription   (Followers: 27, 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: 7)
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: 45, SJR: 3.25, h-index: 43)
Advances in Engineering Software     Hybrid Journal   (Followers: 26, 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: 43, SJR: 5.465, h-index: 64)
Advances in Exploration Geophysics     Full-text available via subscription   (Followers: 3)
Advances in Fluorine Science     Full-text available via subscription   (Followers: 8)
Advances in Food and Nutrition Research     Full-text available via subscription   (Followers: 51, 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 Human Factors/Ergonomics     Full-text available via subscription   (Followers: 26)
Advances in Imaging and Electron Physics     Full-text available via subscription   (Followers: 2, SJR: 0.396, h-index: 27)
Advances in Immunology     Full-text available via subscription   (Followers: 36, SJR: 4.152, h-index: 85)
Advances in Inorganic Chemistry     Full-text available via subscription   (Followers: 9, SJR: 1.132, h-index: 42)
Advances in Insect Physiology     Full-text available via subscription   (Followers: 3, SJR: 1.274, h-index: 27)
Advances in Integrative Medicine     Hybrid Journal   (Followers: 6)
Advances in Intl. Accounting     Full-text available via subscription   (Followers: 4)
Advances in Life Course Research     Hybrid Journal   (Followers: 8, SJR: 0.764, h-index: 15)
Advances in Lipobiology     Full-text available via subscription   (Followers: 2)
Advances in Magnetic and Optical Resonance     Full-text available via subscription   (Followers: 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: 8, 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 Organ Biology     Full-text available via subscription   (Followers: 2)
Advances in Organometallic Chemistry     Full-text available via subscription   (Followers: 15, SJR: 2.885, h-index: 45)
Advances in Parallel Computing     Full-text available via subscription   (Followers: 7, SJR: 0.148, h-index: 11)
Advances in Parasitology     Full-text available via subscription   (Followers: 7, SJR: 2.37, h-index: 73)
Advances in Pediatrics     Full-text available via subscription   (Followers: 24, SJR: 0.4, h-index: 28)
Advances in Pharmaceutical Sciences     Full-text available via subscription   (Followers: 13)
Advances in Pharmacology     Full-text available via subscription   (Followers: 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: 62)
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: 2, SJR: 0.1, h-index: 2)
Advances in Space Biology and Medicine     Full-text available via subscription   (Followers: 5)
Advances in Space Research     Full-text available via subscription   (Followers: 360, 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: 16)
Advances in Veterinary Science and Comparative Medicine     Full-text available via subscription   (Followers: 13)
Advances in Virus Research     Full-text available via subscription   (Followers: 5, SJR: 1.878, h-index: 68)
Advances in Water Resources     Hybrid Journal   (Followers: 44, 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: 331, 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: 417, SJR: 1.385, h-index: 72)
Agri Gene     Hybrid Journal  
Agricultural and Forest Meteorology     Hybrid Journal   (Followers: 16, SJR: 2.18, h-index: 116)
Agricultural Systems     Hybrid Journal   (Followers: 30, SJR: 1.275, h-index: 74)
Agricultural Water Management     Hybrid Journal   (Followers: 40, SJR: 1.546, h-index: 79)
Agriculture and Agricultural Science Procedia     Open Access  
Agriculture and Natural Resources     Open Access   (Followers: 2)
Agriculture, Ecosystems & Environment     Hybrid Journal   (Followers: 55, SJR: 1.879, h-index: 120)
Ain Shams Engineering J.     Open Access   (Followers: 5, SJR: 0.434, h-index: 14)
Air Medical J.     Hybrid Journal   (Followers: 5, SJR: 0.234, h-index: 18)
AKCE Intl. J. of Graphs and Combinatorics     Open Access   (SJR: 0.285, h-index: 3)
Alcohol     Hybrid Journal   (Followers: 11, SJR: 0.922, h-index: 66)
Alcoholism and Drug Addiction     Open Access   (Followers: 8)
Alergologia Polska : Polish J. of Allergology     Full-text available via subscription   (Followers: 1)
Alexandria Engineering J.     Open Access   (Followers: 1, SJR: 0.436, h-index: 12)
Alexandria J. of Medicine     Open Access   (Followers: 1)
Algal Research     Partially Free   (Followers: 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: 9, SJR: 0.158, h-index: 9)
Alzheimer's & Dementia     Hybrid Journal   (Followers: 46, SJR: 4.289, h-index: 64)
Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring     Open Access   (Followers: 4)
Alzheimer's & Dementia: Translational Research & Clinical Interventions     Open Access   (Followers: 4)
Ambulatory Pediatrics     Hybrid Journal   (Followers: 5)
American Heart J.     Hybrid Journal   (Followers: 49, SJR: 3.157, h-index: 153)
American J. of Cardiology     Hybrid Journal   (Followers: 48, SJR: 2.063, h-index: 186)
American J. of Emergency Medicine     Hybrid Journal   (Followers: 40, SJR: 0.574, h-index: 65)
American J. of Geriatric Pharmacotherapy     Full-text available via subscription   (Followers: 9, SJR: 1.091, h-index: 45)
American J. of Geriatric Psychiatry     Hybrid Journal   (Followers: 14, SJR: 1.653, h-index: 93)
American J. of Human Genetics     Hybrid Journal   (Followers: 32, SJR: 8.769, h-index: 256)
American J. of Infection Control     Hybrid Journal   (Followers: 26, SJR: 1.259, h-index: 81)
American J. of Kidney Diseases     Hybrid Journal   (Followers: 32, SJR: 2.313, h-index: 172)
American J. of Medicine     Hybrid Journal   (Followers: 46, 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: 199, SJR: 2.255, h-index: 171)
American J. of Ophthalmology     Hybrid Journal   (Followers: 59, SJR: 2.803, h-index: 148)
American J. of Ophthalmology Case Reports     Open Access   (Followers: 6)
American J. of Orthodontics and Dentofacial Orthopedics     Full-text available via subscription   (Followers: 6, SJR: 1.249, h-index: 88)
American J. of Otolaryngology     Hybrid Journal   (Followers: 25, SJR: 0.59, h-index: 45)
American J. of Pathology     Hybrid Journal   (Followers: 27, SJR: 2.653, h-index: 228)
American J. of Preventive Medicine     Hybrid Journal   (Followers: 25, SJR: 2.764, h-index: 154)
American J. of Surgery     Hybrid Journal   (Followers: 35, SJR: 1.286, h-index: 125)
American J. of the Medical Sciences     Hybrid Journal   (Followers: 12, SJR: 0.653, h-index: 70)
Ampersand : An Intl. J. of General and Applied Linguistics     Open Access   (Followers: 6)
Anaerobe     Hybrid Journal   (Followers: 4, SJR: 1.066, h-index: 51)
Anaesthesia & Intensive Care Medicine     Full-text available via subscription   (Followers: 58, SJR: 0.124, h-index: 9)
Anaesthesia Critical Care & Pain Medicine     Full-text available via subscription   (Followers: 12)
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: 12)
Anesthésie & Réanimation     Full-text available via subscription   (Followers: 1)
Anesthesiology Clinics     Full-text available via subscription   (Followers: 22, SJR: 0.421, h-index: 40)
Angiología     Full-text available via subscription   (SJR: 0.124, h-index: 9)
Angiologia e Cirurgia Vascular     Open Access  
Animal Behaviour     Hybrid Journal   (Followers: 172, SJR: 1.907, h-index: 126)

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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  [3049 journals]
  • Fullerene-PAMAM(G5) composite modified impedimetric biosensor to detect
           Fetuin-A in real blood samples
    • Authors: Zihni Onur Uygun; Çağdaş Şahin; Merve Yılmaz; Yasemin Akçay; Ali Akdemir; Ferhan Sağın
      Pages: 11 - 15
      Abstract: Publication date: 1 February 2018
      Source:Analytical Biochemistry, Volume 542
      Author(s): Zihni Onur Uygun, Çağdaş Şahin, Merve Yılmaz, Yasemin Akçay, Ali Akdemir, Ferhan Sağın
      The aim of this study is to develop a nanomaterial-dendrimer composite modified biosensor to detect Fetuin-A (HFA) in real blood samples. For this purpose, we designed an Electrochemical Impedance Spectroscopy (EIS) based anti-Fetuin-A (Anti-HFA) modified biosensor system and tested in real blood samples. Chronoimpedance was also employed. The same samples were analyzed with ELISA and the results were compared for validation of the new system. Gold screen printed electrodes (AuSPE) were used as transducer. Firstly, a self-assembly monolayer (SAM) was formed on gold surface by 4-aminothiophenol (4-ATP), Fullerene and PAMAM-NH2 (G5), layers were formed, consecutively. Then Anti-HFA was immobilized on Au/4-ATP/Fullerene/PAMAM electrode via glutaraldehyde. The chronoimpedance test was employed to investigate the optimum analysis duration. According to the data of chronoimpedance, the total analysis time for EIS was chosen as 3 min. The new biosensor was compared with the ELISA method which required 150 min. The calibration curve was prepared electron transfer resistance of the electrode (ΔRet) per minute as ohm and 1.66–134 ng/mL.min with a R2 = 0.9912. The LOD and LOQ of the biosensor was calculated as 0.48 ng/mL.min, 1.46 ng/mL.min, respectively. Linear regression analysis indicated that the novel developed biosensor results agreed well with that of the conventional ELISA assay.

      PubDate: 2017-11-30T19:09:06Z
      DOI: 10.1016/j.ab.2017.11.007
      Issue No: Vol. 542 (2017)
  • One-step FPLC-size-exclusion chromatography procedure for purification of
           rDMBT1 6 kb with increased biological activity
    • Authors: Martina Tuttolomondo; Pernille Lund Hansen; Jan Mollenhauer; Henrik J. Ditzel
      Pages: 16 - 19
      Abstract: Publication date: 1 February 2018
      Source:Analytical Biochemistry, Volume 542
      Author(s): Martina Tuttolomondo, Pernille Lund Hansen, Jan Mollenhauer, Henrik J. Ditzel
      Deleted in Malignant Brain Tumor 1 (DMBT1, alias SAG or gp340) is a pattern recognition receptor involved in immune defense, cell polarization, differentiation and regeneration. To investigate the role of the protein in physiological and pathological processes, the protein has often been isolated from saliva or produced in vitro and purified by a multistep affinity purification procedure using bacteria, followed by FPLC. Here, we compared a simple, one-step FPLC-SEC protocol for purification of recombinant DMBT1 6 kb, with that of the standard bacteria affinity purification-based protocol. Our data suggest that our FPLC-SEC protocol yields DMBT1 in a more native conformation.

      PubDate: 2017-11-30T19:09:06Z
      DOI: 10.1016/j.ab.2017.11.015
      Issue No: Vol. 542 (2017)
  • Disulfide bond mapping of Pfs25, a recombinant malaria transmission
           blocking vaccine candidate
    • Authors: Shwu-Maan Lee; Jordan Plieskatt; C. Richter King
      Pages: 20 - 23
      Abstract: Publication date: 1 February 2018
      Source:Analytical Biochemistry, Volume 542
      Author(s): Shwu-Maan Lee, Jordan Plieskatt, C. Richter King
      A liquid chromatography tandem-mass spectrometry method was developed to map the eleven disulfide bonds in Pfs25, a malaria transmission-blocking vaccine candidate. The compact and complex nature of Pfs25 has led to difficulties in prior peptide mapping efforts. Here, we report confirmation of proper disulfide pairing of a recombinant Pfs25, by optimizing denaturation and digestion with trypsin/Lys-C. The digested peptides were separated by reversed phase HPLC to obtain the peptide map and elucidate the disulfide linkages. MSE fragmentation confirmed the digested peptides and disulfide bonds. The eleven disulfide bonds and locations matched the predicted Pvs25 crystal structure, a Pfs25 homologue.
      Graphical abstract image

      PubDate: 2017-11-30T19:09:06Z
      DOI: 10.1016/j.ab.2017.11.009
      Issue No: Vol. 542 (2017)
  • 15N CEST data and traditional model-free analysis capture fast internal
           dynamics of DJ-1
    • Authors: Jonathan Catazaro; Tessa Andrews; Nicole M. Milkovic; Jiusheng Lin; Austin J. Lowe; Mark A. Wilson; Robert Powers
      Pages: 24 - 28
      Abstract: Publication date: 1 February 2018
      Source:Analytical Biochemistry, Volume 542
      Author(s): Jonathan Catazaro, Tessa Andrews, Nicole M. Milkovic, Jiusheng Lin, Austin J. Lowe, Mark A. Wilson, Robert Powers
      Previous studies have shown that relaxation parameters and fast protein dynamics can be quickly elucidated from 15N-CEST experiments [1]. Longitudinal R 1 and transverse R 2 values were reliably derived from fitting of CEST profiles. Herein we show that 15N-CEST experiments and traditional modelfree analysis provide the internal dynamics of three states of human protein DJ-1 at physiological temperature. The chemical exchange profiles show the absence of a minor state conformation and, in conjunction with 1H-15N NOEs, show increased mobility. R 1 and R 2 values remained relatively unchanged at the three naturally occurring oxidation states of DJ-1, but exhibit striking NOE differences. The NOE data was, therefore, essential in determining the internal motions of the DJ-1 proteins. To the authors' knowledge, we present the first study that combines 15N CEST data with traditional model-free analyses in the study of a biological system and affirm that more ‘lean’ model-free approaches should be used cautiously.

      PubDate: 2017-11-30T19:09:06Z
      DOI: 10.1016/j.ab.2017.11.012
      Issue No: Vol. 542 (2017)
  • New spectrophotometric assay for assessments of catalase activity in
           biological samples
    • Authors: Mahmoud Hussein Hadwan; Seenaa kadhum Ali
      Pages: 29 - 33
      Abstract: Publication date: 1 February 2018
      Source:Analytical Biochemistry, Volume 542
      Author(s): Mahmoud Hussein Hadwan, Seenaa kadhum Ali
      A novel, simple, and accurate colorimetric assay was established for assessments of catalase activity in biological fluids and tissues. H2O2 dissociation rates are directly proportional to catalase activity, and the principle of the present assay is based on reactions of ammonium metavanadate with H2O2 under acidic conditions. The resulting reduction of vanadium (V) to vanadium (III) produces a red–orange peroxovanadium complex with absorbance maxima at 452 nm. Biological samples containing catalase were incubated with 50-mM phosphate buffer solution containing 10-mM H2O2 as a substrate for two min. Subsequently, ammonium metavanadate in sulfuric acid was used as an indicator reagent and was added to reaction mixtures to determine remaining H2O2 concentrations. The precision of the present novel assay was indicated by coefficients of variation of 4.09% within runs and 2.56% between runs. Moreover, in experiments with homogenized red blood cell solutions, peroxovanate and dichromate assays of catalase activities were highly correlated (r = 0.993). In further experiments, we demonstrated application of the peroxovanadate method to assessments of catalase activity in bacterial and liver homogenates. The present method is accurate, simple, rapid, and inexpensive and can be used for routine clinical measurements and scientific investigations.
      Graphical abstract image

      PubDate: 2017-11-30T19:09:06Z
      DOI: 10.1016/j.ab.2017.11.013
      Issue No: Vol. 542 (2017)
  • A new look at an old view of denaturant induced protein unfolding
    • Authors: Damien Hall; Akira Kinjo; Yuji Goto
      Pages: 40 - 57
      Abstract: Publication date: 1 February 2018
      Source:Analytical Biochemistry, Volume 542
      Author(s): Damien Hall, Akira Kinjo, Yuji Goto
      We re-examine a site-binding approach independently proposed by Schellman (Schellman, J.A. (1958) Compt. rend. Lab. Carlsberg Ser. Chim. 30, 439–449) and Aune and Tanford (Aune, K.C. and Tanford, D. (1969) Biochemistry, 8, 4586–4590) for explicitly including the denaturant concentration within the protein unfolding equilibrium. We extend and formalize the approach through development of a multi-dimensional analytical model in which the folding reaction coordinate is defined by the number of denaturant molecules bound to sites located on either the initially folded, or unfolded, states of the protein. We use the developed method to re-examine the mechanistic determinants underlying the sigmoidal shape of the unfolding transition. A natural feature of our method is that it presents a landscape picture of the denaturant induced protein unfolding reaction.

      PubDate: 2017-11-30T19:09:06Z
      DOI: 10.1016/j.ab.2017.11.011
      Issue No: Vol. 542 (2017)
  • Development of a low-cost paper-based ELISA method for rapid Escherichia
           coli O157:H7 detection
    • Authors: Bo Pang; Chao Zhao; Li Li; Xiuling Song; Kun Xu; Juan Wang; Yushen Liu; Kaiyue Fu; Hao Bao; Dandan Song; Xiangjun Meng; Xiaofeng Qu; Zhuping Zhang; Juan Li
      Pages: 58 - 62
      Abstract: Publication date: 1 February 2018
      Source:Analytical Biochemistry, Volume 542
      Author(s): Bo Pang, Chao Zhao, Li Li, Xiuling Song, Kun Xu, Juan Wang, Yushen Liu, Kaiyue Fu, Hao Bao, Dandan Song, Xiangjun Meng, Xiaofeng Qu, Zhuping Zhang, Juan Li
      Escherichia coli O157: H7 (E. coli O157: H7) has become one of the most dangerous foodborne pathogenic bacteria around the world. Currently, because of the tedious, high-cost and stringent laboratory conditions required, the conventional E. coli O157: H7 detection methods, such as culture-based methods and polymerase chain reaction (PCR), have much limitation. Thus, we developed a novel paper-based enzyme-linked immunosorbent assay (p-ELISA) with shorter operation duration, lower cost, relatively higher sensitivity and wider application. This method required less than 3 h and 5 μL of sample to complete the detection. The limit of detection (LOD) for E. coli O157: H7 reached 1 × 104 CFU/mL with high specificity. To be more suitable for on-site testing, the readout could be rapidly obtained without any expensive instruments. In this study, we chose E. coli O157:H7 as the representative, and our method could provide a platform for determination of other pathogenic bacteria.

      PubDate: 2017-11-30T19:09:06Z
      DOI: 10.1016/j.ab.2017.11.010
      Issue No: Vol. 542 (2017)
  • Hydrophilic modified magnetic multi-walled carbon nanotube for dispersive
           solid/liquid phase microextraction of sunitinib in human samples
    • Authors: Sara Hooshmand; Zarrin Es'haghi
      Pages: 76 - 83
      Abstract: Publication date: 1 February 2018
      Source:Analytical Biochemistry, Volume 542
      Author(s): Sara Hooshmand, Zarrin Es'haghi
      In this paper, a novel approach for the efficient microextraction and determination of anticancer drug, sunitinib from human samples is described. We synthesized a new nanocomposite; honey coated magnetic multi-walled carbon nanotubes (Honey@magnetic-CNTs). This nanocomposite retains the magnetic properties of individual magnetic nanoparticles (MNPs) and can be effectively separated under an external magnetic field. The synthesized nanoparticles were characterized by FT-IR, VSM, EDAX and XRD and TEM studies. The spherical particles obtained before and after the functionalization had sizes of 14 nm and 16 nm, respectively. The method is based on Honey@magnetic-CNTs assisted dispersive solid-liquid phase microextraction for determination and analysis of the drug. The influences of experimental parameters were investigated. Under optimal conditions, the applied nanocomposite showed good performance, high sensitivity and fast extraction of the analyte from biological samples. In linearity test, the regression correlation coefficient was obtained more than 99% for analyte of interest and linear dynamic range for the proposed method was from 5 to 5000 ng mL−1. Method detection and quantification limits were 1.58 ng mL−1 and 5.28 ng mL−1, respectively. Relative standard deviation was 3.15%.

      PubDate: 2017-11-30T19:09:06Z
      DOI: 10.1016/j.ab.2017.11.019
      Issue No: Vol. 542 (2017)
  • Label-free electrochemical immunoassay for neuron specific enolase based
           on 3D macroporous reduced graphene oxide/polyaniline film
    • Authors: Qi Zhang; Xiaoyan Li; Chunhua Qian; Li Dou; Feng Cui; Xiaojun Chen
      Pages: 1 - 8
      Abstract: Publication date: 1 January 2018
      Source:Analytical Biochemistry, Volumes 540–541
      Author(s): Qi Zhang, Xiaoyan Li, Chunhua Qian, Li Dou, Feng Cui, Xiaojun Chen
      The content of neuron specific enolase (NSE) in serum is considered to be an essential indicator of small cell lung cancer (SCLC). Here, a novel label-free electrochemical immunoassay for the detection of NSE based on the three dimensionally macroporous reduced graphene oxide/polyaniline (3DM rGO/PANI) film has been proposed. The 3DM rGO/PANI film was constructed by electrochemical co-deposition of GO and aniline into the interspaces of a sacrificial silica opal template modified Au slice. During the co-deposition, GO was successfully reduced by aniline and PANI could be deposited on the surfaces of rGO sheets. The ratio of rGO and PANI in the composite was also optimized to achieve the maximum electrochemical performance. The 3DM rGO/PANI composite provided larger specific surface area for the antibody immobilization, exhibited enhanced conductivity for electron transfer, and more important was that PANI acted as the electroactive probe for indicating the NSE concentration. Under the optimal conditions, a linear current response of PANI to NSE concentration was obtained over 0.5 pg mL−1-10.0 ng mL−1 with a detection limit of 0.1 pg mL−1. Moreover, the immunosensor showed excellent selectivity, good stability, satisfactory reproducibility and regeneration, and was employed to detect NSE in clinical serum specimens.
      Graphical abstract image

      PubDate: 2017-11-11T14:30:25Z
      DOI: 10.1016/j.ab.2017.10.009
      Issue No: Vol. 540-541 (2017)
  • Chelatable trace zinc causes low, irreproducible KDAC8 activity
    • Authors: Tasha B. Toro; Samantha A. Edenfield; Brandon J. Hylton; Terry J. Watt
      Pages: 9 - 14
      Abstract: Publication date: 1 January 2018
      Source:Analytical Biochemistry, Volumes 540–541
      Author(s): Tasha B. Toro, Samantha A. Edenfield, Brandon J. Hylton, Terry J. Watt
      Acetylation is an important regulatory mechanism in cells, and emphasis is being placed on identifying substrates and small molecule modulators of this post-translational modification. However, the reported in vitro activity of the lysine deacetylase KDAC8 is inconsistent across experimental setups, even with the same substrate, complicating progress in the field. We detected trace levels of zinc, a known inhibitor of KDAC8 when present in excess, even in high-quality buffer reagents, at concentrations that are sufficient to significantly inhibit the enzyme under common reaction conditions. We hypothesized that trace zinc in solution could account for the observed variability in KDAC8 activity. We demonstrate that addition of chelators, including BSA, EDTA, and citrate, and/or the use of a phosphate-based buffer instead of the more common tris-based buffer, eliminates the inhibition from low levels of zinc as well as the dependence of specific activity on enzyme concentration. This results in high KDAC8 activity that is consistent across buffer systems, even using low concentrations of enzyme. We report conditions that are suitable for several assays to increase both enzyme activity and reproducibility. Our results have significant implications for approaches used to identify substrates and small molecule modulators of KDAC8 and interpretation of existing data.

      PubDate: 2017-11-11T14:30:25Z
      DOI: 10.1016/j.ab.2017.10.024
      Issue No: Vol. 540-541 (2017)
  • An ELISA method to estimate the mono ADP-ribosyltransferase activities:
           e.g in pertussis toxin and vaccines
    • Authors: Catpagavalli Asokanathan; Sharon Tierney; Christina R. Ball; George Buckle; Ami Day; Simon Tanley; Adrian Bristow; Kevin Markey; Dorothy Xing; Chun-Ting Yuen
      Pages: 15 - 19
      Abstract: Publication date: 1 January 2018
      Source:Analytical Biochemistry, Volumes 540–541
      Author(s): Catpagavalli Asokanathan, Sharon Tierney, Christina R. Ball, George Buckle, Ami Day, Simon Tanley, Adrian Bristow, Kevin Markey, Dorothy Xing, Chun-Ting Yuen
      ADP-ribosyltransferase activities have been observed in many prokaryotic and eukaryotic species and viruses and are involved in many cellular processes, including cell signalling, DNA repair, gene regulation and apoptosis. In a number of bacterial toxins, mono ADP-ribosyltransferase is the main cause of host cell cytotoxicity. Several approaches have been used to analyse this biological system from measuring its enzyme products to its functions. By using a mono ADP-ribose binding protein we have now developed an ELISA method to estimate native pertussis toxin mono ADP-ribosyltransferase activity and its residual activities in pertussis vaccines as an example. This new approach is easy to perform and adaptable in most laboratories. In theory, this assay system is also very versatile and could measure the enzyme activity in other bacteria such as Cholera, Clostridium, E. coli, Diphtheria, Pertussis, Pseudomonas, Salmonella and Staphylococcus by just switching to their respective peptide substrates. Furthermore, this mono ADP-ribose binding protein could also be used for staining mono ADP-ribosyl products resolved on gels or membranes.

      PubDate: 2017-11-11T14:30:25Z
      DOI: 10.1016/j.ab.2017.10.025
      Issue No: Vol. 540-541 (2017)
  • Helix structure of the double-stranded DNA for aptameric biosensing and
           imaging of cytochrome c
    • Authors: Somayeh Jamshidi Moghadam; Azadeh Azadbakh
      Pages: 20 - 29
      Abstract: Publication date: 1 January 2018
      Source:Analytical Biochemistry, Volumes 540–541
      Author(s): Somayeh Jamshidi Moghadam, Azadeh Azadbakh
      Here, a method is introduced for construction the aptameric biosensor for biosensing detection of cytochrome C (CYC) based on chain-shape structure of aptasensor by using highly dispersed silver nanoparticles (AgNPs) on acid-oxidized carbon nanotube (CNTs) substrate. The animated capture probe (ssDNA1) and CYC-aptamer (ssDNA2) was immobilized on AgNPs/CNTs surface by covalent amide bonds formed by the carboxyl groups on the nanotubes and the amino groups on the oligonucleotides and hybridization, respectively. In this protocol, the nucleic acids at both ends of the ssDNA1 were sequenced to be complementary (tailor-made ssDNA1). The helix structure of the double-stranded DNA was fabricated by hybridizing ssDNA2 with its complementary sequence (ssDNA1). CYC-aptamer could be forced to dissociate from the sensing interface after CYC triggered structure switching of the aptamer and ssDNA1 thus tend to form a chain-shape structure through the hybridization of the complementary sequences at both its ends. The proposed assay permitted to detect CYC in the linear range of 0.01–750 nM with a very low limit of detection (LOD) (1.66 pM). In addition, the specificity of this sensing system for the detection of CYC was also demonstrated by using albumin, fructose, myoglobin, and hemoglobin.

      PubDate: 2017-11-11T14:30:25Z
      DOI: 10.1016/j.ab.2017.10.016
      Issue No: Vol. 540-541 (2017)
  • Antibody drug quantitation in coexistence with anti-drug antibodies on
           nSMOL bioanalysis
    • Authors: Noriko Iwamoto; Akinobu Hamada; Takashi Shimada
      Pages: 30 - 37
      Abstract: Publication date: 1 January 2018
      Source:Analytical Biochemistry, Volumes 540–541
      Author(s): Noriko Iwamoto, Akinobu Hamada, Takashi Shimada
      Therapeutic monoclonal antibodies (mAbs) are developed for treatment of diverse cancers and autoimmune diseases. For expansion of mAbs approval against unapproved diseases and pharmaceutical development, pharmacokinetics study is very important. Bioanalysis provides one of the most essential index against pharmacokinetics information. So far, we developed useful method for bioanalysis of mAbs in plasma or serum, nSMOL: nano-surface and molecular-orientation limited proteolysis. This method can provide accurate and reproducible value of mAbs content in plasma. Quantification of mAbs using ELISA is strongly influenced by endogenous ligand or anti-drug antibodies. In this report, we exhibited the role of nSMOL proteolysis coupled to LC-MS/MS analysis against quantification of mAbs bound to some binding molecules. The ligands against mAbs do not affect quantification of mAbs concentration in plasma using nSMOL proteolysis. On the other hands, some anti-drug antibodies (ADA), such as idiotypic antibodies, inhibit quantification of mAbs using nSMOL proteolysis. Acid dissociation has some efficacy in accurate value of quantitation of ADA binding mAbs using nSMOL proteolysis coupled to LC-MS/MS analysis. Accordingly, we consider that nSMOL method will contribute to understanding of mAb PK data and therapeutic reference combining with ADA measurements.

      PubDate: 2017-11-18T14:43:28Z
      DOI: 10.1016/j.ab.2017.11.002
      Issue No: Vol. 540-541 (2017)
  • Detection of SNPs of T2DM susceptibility genes by a ligase detection
           reaction–fluorescent nanosphere technique
    • Authors: Yan Chen; Ying Zhao; Yan-Bo Li; Yan-Jun Wang; Gui-Zhen Zhang
      Pages: 38 - 44
      Abstract: Publication date: 1 January 2018
      Source:Analytical Biochemistry, Volumes 540–541
      Author(s): Yan Chen, Ying Zhao, Yan-Bo Li, Yan-Jun Wang, Gui-Zhen Zhang
      Objective To establish a high throughput, low cost, and simple nanotechnology-based method for the detection of single nucleotide polymorphism (SNP) loci in type 2 diabetes mellitus (T2DM). Methods Multiplex ligase detection reaction (LDR) amplification was performed using fluorescently labeled magnetic nanosphere-bound upstream LDR probes and downstream probes labeled with a unique fluorescent group for each SNP locus. The amplified LDR products were separated by magnetic nanospheres and then scanned by fluorescence spectroscopy. Four SNP loci associated with T2DM were detected, including the rs13866634 locus in SLC30A8, rs10811661in CDKN2A/2B, rs1111875 in the HHEX gene, and rs7903146 in the TCF7L2 gene. The SNP genotype was also determined by DNA sequencing as a control. Results The SNP genotypes of the four gene loci determined by the nanosphere-based multiplex LDR method were consistent with the DNA sequencing results. The accuracy rate was 100%. Conclusion A method based on multiplex PCR and LDR was established for simultaneous detection of four SNP loci of T2DM susceptibility genes.

      PubDate: 2017-11-18T14:43:28Z
      DOI: 10.1016/j.ab.2017.11.003
      Issue No: Vol. 540-541 (2017)
  • Functionalization of paramagnetic nanoparticles for protein immobilization
           and purification
    • Authors: Lara A.B.C. Carneiro; Richard J. Ward
      Pages: 45 - 51
      Abstract: Publication date: 1 January 2018
      Source:Analytical Biochemistry, Volumes 540–541
      Author(s): Lara A.B.C. Carneiro, Richard J. Ward
      A paramagnetic nanocomposite coated with chitosan and N-(5-Amino-1-carboxy-pentyl) iminodiacetic acid (NTA) that is suitable for protein immobilization applications has been prepared and characterized. The nanoparticle core was synthesized by controlled aggregation of Fe3O4 under alkaline conditions, and Transmission Electron Microscopy revealed a size distribution of 10–50 nm. The nanoparticle core was coated with chitosan and derivatized with glutaraldehyde and NTA, as confirmed by Fourier Transform Infrared Spectroscopy. The final nanoparticles were used as a metal affinity matrix to separate a recombinant polyhistidine-tagged β-galactosidase from Bacillus subtilis directly from E. coli cell lysates with high purity (>95%). After loading with Ni2+, nanoparticles demonstrated a binding capacity of 250 μg of a polyhistidine-tagged β-galactosidase per milligram of support. The immobilized enzyme retained 80% activity after 9 cycles of washing, and the immobilized recombinant protein could be eluted with high purity with imidazole. The applications for these nanomagnetic composites extend beyond protein purification, and can also be used for immobilizing enzymes, where the β-galactosidase immobilized on the nanomagnetic support was used in multiple cycles of catalytic reactions with no significant loss of catalytic activity.
      Graphical abstract image

      PubDate: 2017-11-30T19:09:06Z
      DOI: 10.1016/j.ab.2017.11.005
      Issue No: Vol. 540-541 (2017)
  • A recombinant fusion protein-based, fluorescent protease assay for high
           throughput-compatible substrate screening
    • Authors: Beáta Bozóki; Lívia Gazda; Ferenc Tóth; Márió Miczi; János András Mótyán; József Tőzsér
      Pages: 52 - 63
      Abstract: Publication date: 1 January 2018
      Source:Analytical Biochemistry, Volumes 540–541
      Author(s): Beáta Bozóki, Lívia Gazda, Ferenc Tóth, Márió Miczi, János András Mótyán, József Tőzsér
      In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection.
      Graphical abstract image

      PubDate: 2017-11-30T19:09:06Z
      DOI: 10.1016/j.ab.2017.11.001
      Issue No: Vol. 540-541 (2017)
  • Using two-site binding models to analyze microscale thermophoresis data
    • Authors: Shih-Chia Tso; Qiuyan Chen; Sergey A. Vishnivetskiy; Vsevolod V. Gurevich; T.M. Iverson; Chad A. Brautigam
      Pages: 64 - 75
      Abstract: Publication date: 1 January 2018
      Source:Analytical Biochemistry, Volumes 540–541
      Author(s): Shih-Chia Tso, Qiuyan Chen, Sergey A. Vishnivetskiy, Vsevolod V. Gurevich, T.M. Iverson, Chad A. Brautigam
      The emergence of microscale thermophoresis (MST) as a technique for determining the dissociation constants for bimolecular interactions has enabled these quantities to be measured in systems that were previously difficult or impracticable. However, most models for analyses of these data featured the assumption of a simple 1:1 binding interaction. The only model widely used for multiple binding sites was the Hill equation. Here, we describe two new MST analytic models that assume a 1:2 binding scheme: the first features two microscopic binding constants (K D(1) and K D(2)), while the other assumes symmetry in the bivalent molecule, culminating in a model with a single macroscopic dissociation constant (K D,M) and a single factor (α) that accounts for apparent cooperativity in the binding. We also discuss the general applicability of the Hill equation for MST data. The performances of the algorithms on both real and simulated data are assessed, and implementation of the algorithms in the MST analysis program PALMIST is discussed.

      PubDate: 2017-11-30T19:09:06Z
      DOI: 10.1016/j.ab.2017.10.013
      Issue No: Vol. 540-541 (2017)
  • Spontaneous luminescence color change in the firefly luciferase assay
    • Authors: Pei-Han Liu; Pawel L. Urban
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Pei-Han Liu, Pawel L. Urban
      The temporal effects of luciferase reaction luminescence have only been discussed in the context of light intensity (flash vs. glow). However, alterations in the color of the light emitted over the course of the luciferase reaction have not been reported. Here, we show a temporal change in the light color emitted during the reaction catalyzed by unmodified firefly luciferase when concentrations of one of the substrates, adenosine triphosphate (ATP), are gradually increased. The temporal color change from green to red occurs within the first few minutes of the luciferase reaction when an ATP-containing solution is either added or synthesized in situ with the aid of an autocatalytic reaction occurring simultaneously. This color change is not accompanied by pH changes. An analysis of the red and green channels demonstrates dissimilar kinetics, suggesting the co-existence of two or more temporally shifted luminescence pathways. The implications of these findings might improve dual-color biosensing/imaging protocols and influence the engineering of biophotonic systems.
      Graphical abstract image

      PubDate: 2017-10-21T21:12:06Z
      DOI: 10.1016/j.ab.2017.10.007
      Issue No: Vol. 539 (2017)
  • 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)
  • Microvalve controlled multi-functional microfluidic chip for divisional
           cell co-culture
    • Authors: Rui Li; Xingjian Zhang; Xuefei Lv; Lina Geng; Yongrui Li; Kuiwei Qin; Yulin Deng
      Pages: 48 - 53
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Rui Li, Xingjian Zhang, Xuefei Lv, Lina Geng, Yongrui Li, Kuiwei Qin, Yulin Deng
      Pneumatic micro-valve controlled microfluidic chip provides precise fluidic control for cell manipulation. In this paper, a multi-functional microfluidic chip was designed for three separate experiments: 1. Different cell lines were dispensed and cultured; 2. Three transfected SH-SY5Y cells were introduced and treated with methyl-phenyl-pyridinium (MPP+) as drug delivery mode; 3. Specific protection and interaction were observed among cell co-culture after nerve damage. The outcomes revealed the potential and practicability of our entire multi-functional pneumatic chip system on different cell biology applications.

      PubDate: 2017-10-21T21:12:06Z
      DOI: 10.1016/j.ab.2017.10.008
      Issue No: Vol. 539 (2017)
  • Isothermal chemical denaturation of large proteins: Path-dependence and
    • Authors: Lucas Wafer; Marek Kloczewiak; Sharon M. Polleck; Yin Luo
      Pages: 60 - 69
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Lucas Wafer, Marek Kloczewiak, Sharon M. Polleck, Yin Luo
      State functions (e.g., ΔG) are path independent and quantitatively describe the equilibrium states of a thermodynamic system. Isothermal chemical denaturation (ICD) is often used to extrapolate state function parameters for protein unfolding in native buffer conditions. The approach is prudent when the unfolding/refolding processes are path independent and reversible, but may lead to erroneous results if the processes are not reversible. The reversibility was demonstrated in several early studies for smaller proteins, but was assumed in some reports for large proteins with complex structures. In this work, the unfolding/refolding of several proteins were systematically studied using an automated ICD instrument. It is shown that: (i) the apparent unfolding mechanism and conformational stability of large proteins can be denaturant-dependent, (ii) equilibration times for large proteins are non-trivial and may introduce significant error into calculations of ΔG, (iii) fluorescence emission spectroscopy may not correspond to other methods, such as circular dichroism, when used to measure protein unfolding, and (iv) irreversible unfolding and hysteresis can occur in the absence of aggregation. These results suggest that thorough confirmation of the state functions by, for example, performing refolding experiments or using additional denaturants, is needed when quantitatively studying the thermodynamics of protein unfolding using ICD.

      PubDate: 2017-11-05T09:50:31Z
      DOI: 10.1016/j.ab.2017.10.001
      Issue No: Vol. 539 (2017)
  • Electrochemical quantification of some water soluble vitamins in
           commercial multi-vitamin using poly-amino acid caped by graphene quantum
           dots nanocomposite as dual signal amplification elements
    • Authors: Nasrin Shadjou; Mohammad Hasanzadeh; Ali Omari
      Pages: 70 - 80
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Nasrin Shadjou, Mohammad Hasanzadeh, Ali Omari
      Rapid analyses of some water soluble vitamins (Vitamin B2, B9, and C) in commercial multi vitamins could be routinely performed in analytical laboratories. This study reports on the electropolymerization of a low toxic and biocompatible polymer “poly aspartic acid-graphene quantum dots” as a novel strategy for surface modification of glassy carbon electrode and preparation a new interface for measurement of selected vitamins in commercial multi vitamins. Electrochemical deposition, as a well-controlled synthesis procedure, has been used for subsequently layer-by-layer preparation of graphene quantum dots nanostructures on a poly aspartic acid using cyclic voltammetry techniques in the regime of −1.5 to 2 V. The field emission scanning electron microscopy indicated immobilization of graphene quantum dots onto poly aspartic acid film. The modified electrode possessed as an effective electroactivity for detection of water soluble vitamins by using cyclic voltammetry, chronoamperometry and differential pulse voltammetry. Enhancement of peak currents is ascribed to the fast heterogeneous electron transfer kinetics that arise from the synergistic coupling between the excellent properties of poly aspartic acid as semiconducting polymer, graphene quantum dots as high density of edge plane sites and chemical modification. Under the optimized analysis conditions, the prepared sensor for detection of VB2, VB9, and VC showed a low limit of quantification 0.22, 0.1, 0.1 μM, respectively.

      PubDate: 2017-11-05T09:50:31Z
      DOI: 10.1016/j.ab.2017.10.011
      Issue No: Vol. 539 (2017)
  • Development of amide-based fluorescent probes for selective measurement of
           carboxylesterase 1 activity in tissue extracts
    • Authors: Sean D. Kodani; Morgane Barthélemy; Shizuo G. Kamita; Bruce Hammock; Christophe Morisseau
      Pages: 81 - 89
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Sean D. Kodani, Morgane Barthélemy, Shizuo G. Kamita, Bruce Hammock, Christophe Morisseau
      Carboxylesterases are well known for their role in the metabolism of xenobiotics. However, recent studies have also implicated carboxylesterases in regulating a number of physiological processes including metabolic homeostasis and macrophage development, underlying the need to quantify them individually. Unfortunately, current methods for selectively measuring the catalytic activity of individual carboxylesterases are not sufficiently sensitive to support many biological studies. In order to develop a more sensitive and selective method to measure the activity of human carboxylesterase 1 (hCE1), we generated and tested novel substrates with a fluorescent aminopyridine leaving group. hCE1 showed at least a 10-fold higher preference for the optimized substrate 4-MOMMP than the 13 other esterases tested. Because of the high stability of 4-MOMMP and its hydrolysis product, this substrate can be used to measure esterase activity over extended incubation periods yielding a low picogram (femtomol) limit of detection. This sensitivity is comparable to current ELISA methods; however, the new assay quantifies only the catalytically active enzyme facilitating direct correlation to biological processes. The method described herein may allow hCE1 activity to be used as a biomarker for predicting drug pharmacokinetics, early detection of hepatocellular carcinoma, and other disease states where the activity of hCE1 is altered.

      PubDate: 2017-11-05T09:50:31Z
      DOI: 10.1016/j.ab.2017.10.014
      Issue No: Vol. 539 (2017)
  • Quantifying variant differences in DNA melting curves: Effects of length,
           melting rate, and curve overlay
    • Authors: M. Li; R.A. Palais; L. Zhou; C.T. Wittwer
      Pages: 90 - 95
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): M. Li, R.A. Palais, L. Zhou, C.T. Wittwer
      High resolution DNA melting of PCR products is a simple technique for sequence variant detection and analysis. However, sensitivity and specificity vary and depend on many factors that continue to be defined. We introduce the area between normalized melting curves as a metric to quantify genotype discrimination. The effects of amplicon size (51–547 bp), melting rate (0.01–0.64 °C/s) and analysis method (curve shape by overlay vs absolute temperature differences) were qualitatively and quantitatively analyzed. To limit experimental variance, we studied a single nucleotide variant with identical predicted wild type and homozygous variant stabilities by nearest neighbor thermodynamic theory. Heterozygotes were easier to detect in smaller amplicons, at faster melting rates, and after curve overlay (superimposition), with some p-values <10−20. As heterozygote melting rates increase, the relative magnitude of heteroduplex contributions to melting curves increases, apparently the result of non-equilibrium processes. In contrast to heterozygotes, the interplay between curve overlay, PCR product size, and analysis method is complicated for homozygote genotype discrimination and is difficult to predict. Similar to temperature cycling in PCR, if the temperature control and temperature homogeneity of the solution are adequate, faster rates improve melting analysis, just like faster rates improve PCR.

      PubDate: 2017-11-05T09:50:31Z
      DOI: 10.1016/j.ab.2017.10.015
      Issue No: Vol. 539 (2017)
  • A multiplex RNA quantification method to determine the absolute amounts of
           mRNA without reverse transcription
    • Authors: Maasa Yokomori; Osamu Gotoh; Yasufumi Murakami; Kenzo Fujimoto; Akira Suyama
      Pages: 96 - 103
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Maasa Yokomori, Osamu Gotoh, Yasufumi Murakami, Kenzo Fujimoto, Akira Suyama
      We have developed a highly sensitive microarray-based method that determines the absolute amounts of mRNA in a total RNA sample in a multiplex manner without reverse transcription. This direct mRNA measurement promotes high-throughput testing and reduces bias in transcriptome analyses. Furthermore, quantification of the absolute amount of mRNA allows transcriptome analysis without common controls or additional, complicated normalization. The method, called Photo-DEAN, was validated using chemically synthesized RNAs of known quantities and mouse liver total RNA samples. We found that the absolute amounts of mRNA were successfully measured without the cDNA synthesis step, with a sensitivity of 15 zmol achieved in 7 h.
      Graphical abstract image

      PubDate: 2017-11-05T09:50:31Z
      DOI: 10.1016/j.ab.2017.10.006
      Issue No: Vol. 539 (2017)
  • Design of titanium nitride- and wolfram carbide-doped RGO/GC electrodes
           for determination of gallic acid
    • Authors: Dalibor M. Stanković; Miloš Ognjanović; Fabian Martin; Ľubomir Švorc; José F.M.L. Mariano; Bratislav Antić
      Pages: 104 - 112
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Dalibor M. Stanković, Miloš Ognjanović, Fabian Martin, Ľubomir Švorc, José F.M.L. Mariano, Bratislav Antić
      In the present paper, the electrochemical behavior and the properties of two modified glassy carbon (GC) electrodes used for quantification of gallic acid in sweet wines were compared. A comparative study was conducted between titanium nitride- or wolfram carbide-doped reduced graphene oxide, labeled as TNrGO and WCrGO, respectively, modified GC electrodes, which are promising composite nanomaterials for electroanalytical applications. For the first time, WCrGO was synthesized and its electroanalytical properties compared with those of TNrGO. Results showed that the proposed materials exhibited enhanced characteristics, e.g., low limits of detection (1.1 μM and 3.1 μM for TNrGO and WCrGO, respectively), wide linear ranges (for TNrGO 4.5–76 μM and for WCrGO 10–100 μM), low adsorption, and low background current, which make them promising candidates for electrochemical sensing applications.

      PubDate: 2017-11-05T09:50:31Z
      DOI: 10.1016/j.ab.2017.10.018
      Issue No: Vol. 539 (2017)
  • A novel voltammetric approach for real-time electrochemical detection of
           targeted nucleic acid sequences using LAMP
    • Authors: Koji Hashimoto; Mika Inada; Keiko Ito
      Pages: 113 - 117
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Koji Hashimoto, Mika Inada, Keiko Ito
      We have developed a novel voltammetric DNA chip for real-time electrochemical detection of targeted nucleic acid sequences using loop-mediated isothermal amplification (LAMP) and ruthenium hexaamine (RuHex) as the intercalative redox compound. A GspSSD DNA polymerase was used for LAMP owing to its tolerance of the intercalative redox compound. The electrochemical reaction of 1 mM RuHex in the LAMP solution was measured continuously by linear sweep voltammetry at 65 °C using an electrochemical DNA chip. According to the LAMP reaction of the positive sample, the cathodic peak current of RuHex increased and the cathodic peak potential of RuHex shifted to negative voltage. The initial number of copies of the targeted nucleic acid was correlated with both the time when the cathodic current began to increase and the time when the cathodic potential began to shift rapidly. 103 to 106 copies/50 μL of the targeted nucleic acid were detected quantitatively and the detection limit was 101 copies within an hour. We expect these results to lead to the realization of simple and stable electrochemical real-time monitoring of targeted nucleic acids while also facilitating the implementation of electrochemical DNA chips in molecular testing.
      Graphical abstract image

      PubDate: 2017-11-05T09:50:31Z
      DOI: 10.1016/j.ab.2017.10.019
      Issue No: Vol. 539 (2017)
  • Immunoaffinity capture coupled with capillary electrophoresis - mass
           spectrometry to study therapeutic protein stability in vivo
    • Authors: Mei Han; Josh T. Pearson; Yunan Wang; Dwight Winters; Marcus Soto; Dan A. Rock; Brooke M. Rock
      Pages: 118 - 126
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Mei Han, Josh T. Pearson, Yunan Wang, Dwight Winters, Marcus Soto, Dan A. Rock, Brooke M. Rock
      Protein engineering is at an all-time high in biopharmaceutics. As a result, absorption, distribution, metabolism and excretion (ADME) of proteins has become more important to understand in the context of engineering strategies to optimize therapeutic properties of potential lead constructs. Immunoaffinity capture coupled with a newly developed capillary electrophoresis – mass spectrometry (CE-MS) system was used to characterize intact protein mass analysis of a wild type Fc-FGF21 construct and a sequence re-engineered Fc-FGF21 construct from an in vivo study. A number of truncated forms were observed and the time courses of the various proteolytic products were identified and compared between the two constructs. The abundances of the intact and truncated forms were used to provide the basis to semi-quantify ADME properties of the two protein forms. The use of this immunoaffinity capture followed by CE-MS based intact mass analysis workflow provided a qualitative and quantitative analysis of the pharmacokinetic profiles of the two proteins. The platform presented here holds great potential in characterization of the ADME properties of proteins.

      PubDate: 2017-11-05T09:50:31Z
      DOI: 10.1016/j.ab.2017.10.005
      Issue No: Vol. 539 (2017)
  • In situ surface protein conjugation of small molecules for SPR
    • Authors: Yijing Wang; Ashton Partridge; Yinqiu Wu
      Pages: 149 - 151
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Yijing Wang, Ashton Partridge, Yinqiu Wu
      Solution based protein conjugation of small molecules involves multiple steps of chemical syntheses, bio-conjugation and purifications which are labor intensive and time-consuming. Since many small molecules have limited water solubility, conjugation to a protein is also a relatively low efficiency process in aqueous solutions. In this study, a model in situ protein conjugation of small molecules was achieved onto SPR surfaces, using progesterone and ovalbumin as a model small-molecule-protein system. The in situ protein conjugation not only eliminated the requirement for wet chemistry, but also provided an easy way to optimize the assay performance and screen various molecular linkers.

      PubDate: 2017-11-05T09:50:31Z
      DOI: 10.1016/j.ab.2017.10.017
      Issue No: Vol. 539 (2017)
  • Fluorometric determination of d-lactate in biological fluids
    • Authors: Torben Larsen
      Pages: 152 - 157
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Torben Larsen
      Objective D-lactic acid in the mammalian body is mainly of microbiological origin and is often located somewhere along the digestive tract. Surgical, extensive re-sectioning of the small bowel may be one of the risk factors for altered balance in the microbiological environment. Higher levels in the body may lead to D-lactate acidosis and neurotoxicity; consequently, the possibility of diagnosis of this condition is important. Several analytical procedures for D-lactate have been introduced, but it is absolutely mandatory to distinguish this metabolite from the much more abundant and naturally occurring stereoisomer L-lactate. If enzymatic analytical methods are used, it is consequently essential to eliminate the response from L-lactate and the ubiquitous enzyme L-lactate dehydrogenase (L-LDH) (and other oxido-reductases) which will interfere with the D-lactate determination heavily. Design and methods The present paper introduces an enzymatic-fluorometric method for determination of D-lactate in biological matrices, including blood plasma, serum and urine. Macro molecules, including enzymes, were initially precipitated by ethanol and the supernatant used for analyses. Several plasma samples were analysed with and without standard addition of both L- and D-lactate in order to validate the assay. Results and conclusions The procedure effectively eliminates enzyme activities that may interfere with the D-lactate quantification, resulting in the situation that L-lactate in the sample does not interfere with the determination. Intra- and inter-assay precision, accuracy and recovery of the analyte were investigated and everything suggests that this method will be acceptable for analytical as well as descriptive purposes. The analytical procedure is suitable for a semi-automated large scale set-up in the laboratory.

      PubDate: 2017-11-05T09:50:31Z
      DOI: 10.1016/j.ab.2017.10.026
      Issue No: Vol. 539 (2017)
  • Glycan profile of CHO derived IgM purified by highly efficient single step
           affinity chromatography
    • Authors: Julia Hennicke; Anna Maria Lastin; David Reinhart; Clemens Grünwald-Gruber; Friedrich Altmann; Renate Kunert
      Pages: 162 - 166
      Abstract: Publication date: 15 December 2017
      Source:Analytical Biochemistry, Volume 539
      Author(s): Julia Hennicke, Anna Maria Lastin, David Reinhart, Clemens Grünwald-Gruber, Friedrich Altmann, Renate Kunert
      Immunoglobulin M (IgM) antibodies are reckoned as promising tools for therapy and diagnostic approaches. Nevertheless, the commercial success of IgMs is hampered due to bottlenecks in recombinant production and downstream processing. IgMs are large, complex and highly glycosylated proteins that are only stable in a limited range of conditions. To investigate these sensitive IgM antibodies we optimized the elution conditions for a commercially available IgM affinity matrix (CaptureSelect™). Applying a small-scale screening system, we optimized our single step purification strategy for high purity, high yield and retained antigen binding capacity. Here we show that IgMs are sensitive to aggregation at very acidic conditions (pH ≤ 3.0) despite often being used for affinity chromatography. We combined pH 3.5 with a high salt concentration to prevent aggregation during elution. The elution strategy presented in this paper will improve IgM processes for further applications. The herein used IgMs were produced in Chinese hamster ovary (CHO) cells. We present the first detailed glycan analysis of IgM produced in CHO cells with predominantly complex type structures at Asn171, Asn332 and Asn395 and oligomannosidic structures at Asn402 and Asn563 similar to human serum-IgM.

      PubDate: 2017-11-05T09:50:31Z
      DOI: 10.1016/j.ab.2017.10.020
      Issue No: Vol. 539 (2017)
  • Circulating tumoral DNA: Preanalytical validation and quality control in a
           diagnostic laboratory
    • Abstract: Publication date: 1 February 2018
      Source:Analytical Biochemistry, Volume 542
      Author(s): Sergey Nikolaev, Laure Lemmens, Thibaud Koessler, Jean-Louis Blouin, Thierry Nouspikel
      We present the results of our technical validation process in establishing the analysis of circulating tumor DNA (ctDNA) as a diagnostic tool. Like most cells in our body, tumor cells shed DNA in the blood flow. Analysis of ctDNA mutational content can provide invaluable information on the genetic makeup of a tumor, and assist oncologists in deciding on therapy, or in following residual disease. However, low absolute amounts of circulating DNA and low tumor fraction constitute formidable analytical challenges. A key step is to avoid contamination with genomic DNA from cell lysis. Several brands of specialized blood collection tubes are available to prevent leukocyte lysis. We show that they are not equally efficient, depending on storage temperature and time before plasma preparation. We report our analysis of preanalytical factors pertaining to ctDNA analysis (tubes, transportation time, temperature) and our conclusions in terms of instructions to prescribing physicians. We also stress the importance of proper DNA quality control and compare several methods, including a differential amplicon length PCR technique which allows determination of multiple QC parameters from minimal amounts of DNA. Altogether, these data provide useful practical information to diagnostic laboratories wishing to implement the assay of ctDNA in clinical practice.

      PubDate: 2017-12-11T12:59:26Z
  • A high-throughput screening campaign to identify inhibitors of DXP
           reductoisomerase (IspC) and MEP cytidylyltransferase (IspD)
    • Abstract: Publication date: 1 February 2018
      Source:Analytical Biochemistry, Volume 542
      Author(s): Amanda Haymond, Tyrone Dowdy, Chinchu Johny, Claire Johnson, Haley Ball, Allyson Dailey, Brandon Schweibenz, Karen Villarroel, Richard Young, Clark J. Mantooth, Trishal Patel, Jessica Bases, Cynthia S. Dowd, Robin D. Couch
      The rise of antibacterial resistance among human pathogens represents a problem that could change the landscape of healthcare unless new antibiotics are developed. The methyl erythritol phosphate (MEP) pathway represents an attractive series of targets for novel antibiotic design, considering each enzyme of the pathway is both essential and has no human homologs. Here we describe a pilot scale high-throughput screening (HTS) campaign against the first and second committed steps in the pathway, catalyzed by DXP reductoisomerase (IspC) and MEP cytidylyltransferase (IspD), using compounds present in the commercially available LOPAC1280 library as well as in an in-house natural product extract library. Hit compounds were characterized to deduce their mechanism of inhibition; most function through aggregation. The HTS workflow outlined here is useful for quickly screening a chemical library, while effectively identifying false positive compounds associated with assay constraints and aggregation.

      PubDate: 2017-12-11T12:59:26Z
  • Contact lens to measure individual ion concentrations in tears and
           applications to dry eye disease
    • Abstract: Publication date: 1 February 2018
      Source:Analytical Biochemistry, Volume 542
      Author(s): Ramachandram Badugu, Bennie H. Jeng, E. Albert Reece, Joseph R. Lakowicz
      Dry eye disease (DED) affects millions of individuals in the United States and worldwide, and the incidence is increasing with an aging population. There is widespread agreement that the measurement of total tear osmolarity is the most reliable test, but this procedure provides only the total ionic strength and does not provide the concentration of each ionic species in tears. Here, we describe an approach to determine the individual ion concentrations in tears using modern silicone hydrogel (SiHG) contact lenses. We made pH (or H3O+, hydronium cation,/OH−, hydroxyl ion) and chloride ion (two of the important electrolytes in tear fluid) sensitive SiHG contact lenses. We attached hydrophobic C18 chains to water-soluble fluorescent probes for pH and chloride. The resulting hydrophobic ion sensitive fluorophores (H-ISF) bind strongly to SiHG lenses and could not be washed out with aqueous solutions. Both H-ISFs provide measurements which are independent of total intensity by use of wavelength-ratiometric measurements for pH or lifetime-based sensing for chloride. Our approach can be extended to fabricate a contact lens which provides measurements of the six dominant ionic species in tears. This capability will be valuable for research into the biochemical processes causing DED, which may improve the ability to diagnose the various types of DED.
      Graphical abstract image

      PubDate: 2017-12-11T12:59:26Z
  • Detection of simultaneous multi-mutations using base-quenched probe
    • Abstract: Publication date: Available online 9 December 2017
      Source:Analytical Biochemistry
      Author(s): Huihui Mao, Guanghua Luo, Jun Zhang, Ning Xu
      The base-quenched probe method for detecting single nucleotide polymorphisms (SNPs) relies on real-time PCR and melting-curve approaches. Here, we applied the most common commercial fluorophores including FAM, HEX, CY5, CY3, TET, JOE, Texas Red and ROX for labeling probes to detect multi-mutations simultaneously according to the different fluorescence channels. Accuracy of the method was confirmed by direct sequencing. The results demonstrated that all above dyes could be influenced by bases and could be applied to detect SNPs. Furthermore, this method was applied to detect APOM rs707921, APOM rs707922 and MCP-1 rs1024611 simultaneously, which was demonstrated successfully.

      PubDate: 2017-12-11T12:59:26Z
  • New Fpg probe chemistry for direct detection of recombinase polymerase
           amplification on lateral flow strips
    • Abstract: Publication date: Available online 9 December 2017
      Source:Analytical Biochemistry
      Author(s): Michael L. Powell, Frank R. Bowler, Aurore J. Martinez, Catherine J. Greenwood, Niall Armes, Olaf Piepenburg
      Rapid, cost-effective and sensitive detection of nucleic acids has the ability to improve upon current practices employed for pathogen detection in diagnosis of infectious disease and food testing. Furthermore, if assay complexity can be reduced, nucleic acid amplification tests could be deployed in resource-limited and home use scenarios. In this study, we developed a novel Fpg (Formamidopyrimidine DNA glycosylase) probe chemistry, which allows lateral flow detection of amplification in undiluted recombinase polymerase amplification (RPA) reactions. The prototype nucleic acid lateral flow chemistry was applied to a human genomic target (rs1207445), Campylobacter jejuni 16S rDNA and two genetic markers of the important food pathogen E. coli O157:H7. All four assays have an analytical sensitivity between 10 and 100 copies DNA per amplification. Furthermore, the assay is performed with fewer hands-on steps than using the current RPA Nfo lateral flow method as dilution of amplicon is not required for lateral flow analysis. Due to the simplicity of the workflow, we believe that the lateral flow chemistry for direct detection could be readily adapted to a cost-effective single-use consumable, ideal for use in non-laboratory settings.
      Graphical abstract image

      PubDate: 2017-12-11T12:59:26Z
  • Studies on the development of antibodies for the highly hydrophobic
           plasticizers DINCH and DEHT
    • Abstract: Publication date: Available online 9 December 2017
      Source:Analytical Biochemistry
      Author(s): Stefanie Baldofski, Carsten Jörg Canitz, Leif-Alexander Garbe, Rudolf J. Schneider
      Diisononylcyclohexane-1,2-dicarboxylate (DINCH) and di-2-ethylhexyl terephthalate (DEHT), two of the most important substitutes for phthalate plasticizers, are used for a wide range of applications. Consequently, an increasing occurrence in urine and environmental samples is reported. Reliable and fast analytical methods for the quantification of these plasticizers are needed. So far, mainly GC-MS or LC-MS methods are used. We aimed to develop the first antibodies and immunoassays allowing for high-throughput analysis of samples. We designed two DINCH hapten structures and one DEHT hapten structure and employed hapten-protein conjugates for the immunization of rabbits. Sensitive competitive enzyme-linked immunosorbent assays (ELISAs) against each hapten using the produced polyclonal antibodies were established. Yet, binding of DINCH to the respective antibodies was not be observed in neither direct nor indirect assay formats, even when using protein conjugates with the heterologous haptens and different carrier proteins in the indirect format. The use of surfactants and solvents in the sample buffer did not result in recognition of the plasticizers. Also, no binding of DEHT in ELISA employing the respective antibodies was detected. We speculate that the production of antibodies against these highly hydrophobic molecules is not possible via our route, however a different hapten design could overcome this obstacle.
      Graphical abstract image

      PubDate: 2017-12-11T12:59:26Z
  • Facile detection of microRNA based on phosphorescence resonance energy
           transfer and duplex-specific nuclease-assisted signal amplification
    • Authors: Jia-jia Yang; Zhi-feng Zhang; Gui-qin Yan
      Abstract: Publication date: Available online 28 October 2017
      Source:Analytical Biochemistry
      Author(s): Jia-jia Yang, Zhi-feng Zhang, Gui-qin Yan
      MicroRNAs (miRNAs) play an important role in many biological processes, and its level in plasma and other biological fluids is closely related to many diseases. In this work, a selective room-temperature phosphorescence (RTP) detection method for miRNA was developed based on a duplex-specific nuclease (DSN) -assisted signal amplification strategy and phosphorescence resonance energy transfer (PRET) between poly-diallyldimethylammonium chloride-modified quantum dots (QDs@PDDA) and 6-carboxy-X-rhodamine-modified miRNA sequences complementary oligonucleotide (ROX-ssDNA). The positively charged QDs@PDDA could adsorb negatively charged ROX-ssDNA by electrostatic interaction, whereas the RTP signal of QDs@PDDA could be efficiently quenched by ROX-ssDNA via PRET. In the presence of microRNA-21 (miR-21) and DSN, miR-21 hybridized with ROX-ssDNA initially to form a DNA-RNA heteroduplex as the substrate of DSN, then ssDNA in DNA-RNA heteroduplex would be cleaved into small fragments by DSN and liberate miR-21 to hybridize with another ROX-ssDNA. Eventually, due to weak interaction between ROX-ssDNA fragments and QDs@PDDA, PRET efficiency continually decreased whereas the RTP signal was significantly amplified. By employing the strategy above, quantitative detection of miR-21 in the range of 0.25–40 nM with a detection limit of 0.16 nM was realized, showing excellent performance with simplicity, good selectivity and the ability to be a promising method for miRNA detection.

      PubDate: 2017-10-29T09:33:23Z
      DOI: 10.1016/j.ab.2017.10.021
  • Detection of Pseudomonas aeruginosa biomarkers from thermally injured mice
           in situ using imaging mass spectrometry
    • Authors: Timothy Hamerly; Jake A. Everett; Nina Paris; Steve T. Fisher; Arivarasan Karunamurthy; Garth A. James; Kendra P. Rumbaugh; Daniel D. Rhoads; Brian Bothner
      Abstract: Publication date: Available online 28 October 2017
      Source:Analytical Biochemistry
      Author(s): Timothy Hamerly, Jake A. Everett, Nina Paris, Steve T. Fisher, Arivarasan Karunamurthy, Garth A. James, Kendra P. Rumbaugh, Daniel D. Rhoads, Brian Bothner
      Monitoring patients with burn wounds for infection is standard practice because failure to rapidly and specifically identify a pathogen can result in poor clinical outcomes, including death. Therefore, a method that facilitates detection and identification of pathogens in situ within minutes of biopsy would be a significant benefit to clinicians. Mass spectrometry is rapidly becoming a standard tool in clinical settings, capable of identifying specific pathogens from complex samples. Imaging mass spectrometry (IMS) expands the information content by enabling spatial resolution of biomarkers in tissue samples as in histology, without the need for specific stains/antibodies. Herein, a murine model of thermal injury was used to study infection of burn tissue by Pseudomonas aeruginosa. This is the first use of IMS to detect P. aeruginosa infection in situ from thermally injured tissue. Multiple molecular features could be spatially resolved to infected or uninfected tissue. This demonstrates the potential use of IMS in a clinical setting to aid doctors in identifying both presence and species of pathogens in tissue.

      PubDate: 2017-10-29T09:33:23Z
      DOI: 10.1016/j.ab.2017.10.012
  • Development of a quantitative immuno-polymerase chain reaction assay to
           detect and quantify low levels of human thyroid stimulating hormone
    • Authors: J.E. Abud; C.G. E.H. Luque H.A. Rodriguez
      Abstract: Publication date: Available online 27 October 2017
      Source:Analytical Biochemistry
      Author(s): J.E. Abud, C.G. Santamaría, E.H. Luque, H.A. Rodriguez
      In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and a highly sensitive immuno-polymerase chain reaction (IPCR) assay specific for detection of human thyroid stimulating hormone (hTSH). Several anti-hTSH monoclonal antibodies (MAbs) were generated using hybridoma technology. Two pairs of MAbs (B-4 and B-9) were rationally selected and the optimal assay conditions of sandwich ELISAs were established. The ELISA prototypes were evaluated with standards calibrated with WHO 2nd International Reference Preparation for hTSH and in comparison with a commercial ELISA Kit. Although the limit of detection (LOD) was 0.1 μIU/ml in all cases, B-9-ELISA showed an analytical performance similar to commercial ELISA Kit. Therefore, we selected the B-9 ELISA to develop a hTSH-IPCR assay applying an “Universal-IPCR” format in standard PCR tubes without pretreatment. The signal amplification was achieved through the interaction between the biotinylated detection MAb and mono-biotinylated DNA probe pre-self-assembled with neutravidin. The hTSH-IPCR assay showed a significant increase in terms of the slope definition of sensitivity in low levels range. Our results support the potential of IPCR technique for being applied in clinical diagnosis of thyroid states.

      PubDate: 2017-10-29T09:33:23Z
  • Quantification of sulphur amino acids by ultra-high performance liquid
           chromatography in aquatic invertebrates
    • Authors: Jennifer C. Thera; Karen A. Kidd; M. Elaine Dodge-Lynch; Robert F. Bertolo
      Abstract: Publication date: Available online 26 October 2017
      Source:Analytical Biochemistry
      Author(s): Jennifer C. Thera, Karen A. Kidd, M. Elaine Dodge-Lynch, Robert F. Bertolo
      We examined the performance of an ultra-high performance liquid chromatography method to quantify protein-bound sulphur amino acids in zooplankton. Both cysteic acid and methionine sulfone were linear from 5 to 250 pmol (r2 = 0.99), with a method detection limit of 13 pmol and 9 pmol, respectively. Although there was no matrix effect on linearity, adjacent peaks and co-eluting noise from the invertebrate proteins increased the detection limits when compared to common standards. Overall, performance characteristics were reproducible and accurate, and provide a means for quantifying sulphur amino acids in aquatic invertebrates, an understudied group.

      PubDate: 2017-10-29T09:33:23Z
      DOI: 10.1016/j.ab.2017.10.022
  • 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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