title: Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection.
abstract: BACKGROUND
  Rapid diagnosis for time-sensitive illnesses such as stroke, cardiac arrest, and septic shock is essential for successful treatment. Much attention has therefore focused on new strategies for rapid and objective diagnosis, such as Point-of-Care Tests (PoCT) for blood biomarkers. Here we use a biomimicry-based approach to demonstrate a new diagnostic platform, based on enzymes tethered to nanoparticles (NPs). As proof of principle, we use oriented immobilization of pyruvate kinase (PK) and luciferase (Luc) on silica NPs to achieve rapid and sensitive detection of neuron-specific enolase (NSE), a clinically relevant biomarker for multiple diseases ranging from acute brain injuries to lung cancer. We hypothesize that an approach capitalizing on the speed and catalytic nature of enzymatic reactions would enable fast and sensitive biomarker detection, suitable for PoCT devices.
  
  
  RESULTS
  We performed in-vitro, animal model, and human subject studies. First, the efficiency of coupled enzyme activities when tethered to NPs versus when in solution was tested, demonstrating a highly sensitive and rapid detection of physiological and pathological concentrations of NSE. Next, in rat stroke models the enzyme-based assay was able in minutes to show a statistically significant increase in NSE levels in samples taken 1 hour before and 0, 1, 3 and 6 hours after occlusion of the distal middle cerebral artery. Finally, using the tethered enzyme assay for detection of NSE in samples from 20 geriatric human patients, we show that our data match well (r = 0.815) with the current gold standard for biomarker detection, ELISA-with a major difference being that we achieve detection in 10 minutes as opposed to the several hours required for traditional ELISA.
  
  
  CONCLUSIONS
  Oriented enzyme immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes. Together, our findings provide proof of concept for using oriented immobilization of active enzymes on NPs as the basis for a highly rapid and sensitive biomarker detection platform. This addresses a key challenge in developing a PoCT platform for time sensitive and difficult to diagnose pathologies.
authors.affiliation: Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Hungerford Hill Rd., Ithaca, NY 14853, United States of America.
authors.forename: Roy
authors.initials: R
authors.lastname: Cohen
authors.affiliation: Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Hungerford Hill Rd., Ithaca, NY 14853, United States of America.|Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, United States of America.
authors.forename: James P
authors.initials: JP
authors.lastname: Lata
authors.affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, United States of America.
authors.forename: Yurim
authors.initials: Y
authors.lastname: Lee
authors.affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, United States of America.
authors.forename: Jean C Cruz
authors.initials: JC
authors.lastname: Hernández
authors.affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, United States of America.
authors.forename: Nozomi
authors.initials: N
authors.lastname: Nishimura
authors.affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, United States of America.
authors.forename: Chris B
authors.initials: CB
authors.lastname: Schaffer
authors.affiliation: Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Hungerford Hill Rd., Ithaca, NY 14853, United States of America.
authors.forename: Chinatsu
authors.initials: C
authors.lastname: Mukai
authors.affiliation: Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Hungerford Hill Rd., Ithaca, NY 14853, United States of America.
authors.forename: Jacquelyn L
authors.initials: JL
authors.lastname: Nelson
authors.affiliation: Central New York ADAC, SUNY Upstate Medical University, Syracuse, NY 13210, United States of America.
authors.forename: Sharon A
authors.initials: SA
authors.lastname: Brangman
authors.affiliation: New York Presbyterian Hospital-Cornell Campus, Cornell University, Ithaca, NY 10065, United States of America.
authors.forename: Yash
authors.initials: Y
authors.lastname: Agrawal
authors.affiliation: Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Hungerford Hill Rd., Ithaca, NY 14853, United States of America.|Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, United States of America.|Atkinson Center for a Sustainable Future, Cornell University, Ithaca, NY 14853, United States of America.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Travis
mesh_terms: D000375:Aging / Q000097:blood*; D000818:Animals; D001681:Biological Assay / Q000592:standards*; D015415:Biomarkers / Q000097:blood; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005260:Female; D017930:Genes, Reporter; D006801:Humans; D020244:Infarction, Middle Cerebral Artery / Q000097:blood* / Q000175:diagnosis / Q000503:physiopathology; D008156:Luciferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008297:Male; D053758:Nanoparticles / Q000737:chemistry; D010751:Phosphopyruvate Hydratase / Q000097:blood*; D019095:Point-of-Care Systems; D011770:Pyruvate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry; D020521:Stroke / Q000097:blood* / Q000175:diagnosis / Q000503:physiopathology; D013997:Time Factors
chemical_list: D015415:Biomarkers; D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D012822:Silicon Dioxide; D008156:Luciferases; D011770:Pyruvate Kinase; D010751:Phosphopyruvate Hydratase