Article pubs.acs.org/ac
Nanoparticle−Drug Bioconjugate as Dual Functional Affinity Ligand for Rapid Point-of-Care Detection of Endotoxin in Water and Serum Prasanta Kalita,#,‡ Anshuman Dasgupta,#,†,‡ Venkataraman Sritharan,§ and Shalini Gupta*,# #
Department of Chemical Engineering, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India Department of Molecular Diagnostics & Biomarkers, Global Medical Education and Research Foundation, Hyderabad 500004, India
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S Supporting Information *
ABSTRACT: Endotoxin or lipopolysaccharide (LPS) is a major constituent of the Gram-negative bacterial cell wall that causes a life-threatening disorder called septicemia resulting from the unregulated activation of the innate immune system. We demonstrate a simple and robust drug-assisted dot blot bioassay for endotoxin detection that can be used right by the critically ill patients’ bedside. Target LPS molecules are trapped from serum or water on glass substrates via longchain alkyls and tagged with reporter gold nanoparticles (NPs) preconjugated to an antibiotic drug called polymyxin B sulfate (PMB). A post-silver-enhancement step enables signal visibility to the bare eye over a wide and clinically relevant concentration range of 50 fg/mL−50 ng/mL, allowing effortless diagnosis of sepsis at various stages, from early sepsis to septic shock.
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Effectiveness of infection control procedures including intervention with antibiotics can rapidly decrease after the onset of sepsis. Therefore, biomarkers that can identify individuals at the risk of sepsis at the presymptomatic stage or at very early stages of infection (first 1 to 2 h), irrespective of the previous antibiotic treatment, can dramatically reduce morbidity and mortality in patients. This may further reduce the development of drug resistance, toxicity, and deterioration of a patient’s condition, which occur due to the empirical antibiotic therapy initiated by the clinician. The only USFDA approved kit for LPS detection in clinical use since 2010 is an endotoxin activity assay (EAA) developed by Spectral Diagnostics, Canada, which measures the natural oxidative burst in neutrophils in blood induced by endotoxin.11−13 This test is, however, prohibitive in cost and requires dedicated chemiluminescence equipment. There are other formats of endotoxin assays available like the dye, radiometric, and ELISA methods, which claim that endotoxin can be tested in biological fluids but none of them have been validated or approved for clinical use yet.14,12 Moreover, they are expensive and tedious to perform with turnaround times of 3 to 4 h and are prone to interference by several contaminants and compounds present in biological samples. The most commonly used diagnostic test is the chromogenic Limulus amebocyte lysate (LAL) assay that is based on the ability of endotoxin to induce coagulation of the hemo lymph of the horseshoe crab, Limulus polyphemus.15,16 This assay is widely used to detect endotoxin contamination in
epticemia is caused by the overwhelming response of the immune system to a microbial insult.1 The main causative agents of sepsis are Gram-negative bacteria due to their predominance in the normal intestinal flora and the environment.2 Mortality rates due to sepsis are highest in wounded soldiers, neonates, and postsurgical patients because of lack of reliable rapid methods for detecting the onset and measuring the progression of systemic infection. We do not have a comprehensive collection of Indian national data regarding the burden due to sepsis nor a cost to the government, but it is estimated that India has about 70 000 critical care beds, whereas more than 2 million beds are actually needed.3 In the United States alone, sepsis costs $16.7 billion and leads to 200 000 deaths annually.4,5 For patients suspected of sepsis, therapeutic decisions are often made on the basis of signs and clinical symptoms without objective evidence of infection or endotoxemia. For instance, diagnosis by physicians relies heavily on overt clinical symptoms of illness like temperature, blood pressure, and heart rate that are neither specific nor fully expressed because of many intensive care unit (ICU) patients being unconscious and immunocompromised due to the ongoing treatment regimens such as a ventilator or ionotropic support. Clinical observations are only supported by the total white blood cell count, toxic granules in neutrophils, and C-reactive protein measurements, all of which are neither sensitive nor specific.6,7 In addition, culture reports are frequently “no growth” due to the ongoing broad-spectrum antibiotic treatment (sensitivity is
