paricalcitol We report a new electrochemical

We report a new electrochemical biosensor for CRP detection as part of a first step in the development of simple, multi-electrode microfluidic clinical diagnostic tools. One of the biggest challenges with multi-analyte electrochemical biosensors is localization of the affinity agents on individual electrodes. One elegant solution to this problem is the use of DNA hybridization to direct localization of paricalcitol or other affinity agents [41]. These techniques, referred to as DNA Zipcoding or DNA-Directed Immobilization (DDI) have been widely used with traditional colorimetric or fluorescence detection but not with electrochemical immunoassays. This report detailing the development of an electrochemical DNA immunoassay (eDI), is made possible by selective modification of Au microwires in an electrochemical cell. The use of microwire electrodes for electrochemical detection in conjunction with paper-based analytical devices has been reported recently [42]. Au is used because it is easily modified with thiolated DNA [43,44]. Once modified, the electrode can be readily blocked to reduce non-specific interactions using mercaptohexanol (MCH) [44–46]. After modification with the ssDNA, an antibody modified with complementary ssDNA is added, resulting in an electrode modified with the target antibody. Finally, binding is measured using Electrochemical Impedance Spectroscopy (EIS). EIS is a common electrochemical technique capable of quantifying surface binding [47,48]. EIS has been used to detect biomolecular events including protein–protein interactions [49,50], DNA hybridization [51–53], DNA–RNA [54] and DNA-protein interactions [55–58].
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Introduction Bacterial infection is a frequent complication among trauma and surgical patients, both civilian and military. Damage to soft tissues and organs with accompanying immune system suppression enables opportunistic pathogens to colonize and infect [36]. Patients with infection are more likely to be admitted to an ICU, require extensive debridement, have longer lengths of stay in hospital, and have a higher risk of early mortality [16,21,44]. Adverse outcomes within one year after injury with accompanying infection include increased risk of death and repeat hospitalization, greater dependence on healthcare, decreased likelihood to return to work, and diminished overall function [15]. According to the World Health Organization, the prevalence of all healthcare associated infections is 4.5% in the United States, 7.1% in Europe, and 15.5% in underdeveloped countries [2]. The rate of infectious complications in the United States military is approximately 35% for combat casualties [33]. The limitations of current standards of diagnosis can lead to delayed interventions and poor prognosis. Military trauma patients in transit to higher echelons of care present a challenge to healthcare providers due to the disparity in technology available to caregivers outside of the hospital setting [39]. Colonization of combat casualties by infectious pathogens increases during transit through evacuation chains [25] and requires reevaluation at each level of care. Culture-based techniques for standard diagnosis take between 24 and 48h and require plating, growth, and examination of colony morphology or color to identify bacteria [31]. The exceedingly long turnaround times required for culture-based methods led to the development of DNA- and enzyme-based assays utilizing nucleic acid and antibody probes for identification and quantification of target bacterial cells. Diagnosis using traditional polymerase chain reaction (PCR) requires technical expertise and sophisticated laboratory equipment. Furthermore, assays with PCR typically require species and/or strain specific probes that may or may not be available for a particular organism. The instability of DNA and antibodies in harsh environments can limit their applicability for use outside of controlled settings.