Method for Low Nanomolar Concentration Analyte Sensing Using Electrochemical Enzymatic Biosensors

We introduce a new electrochemical measurement method compatible with an enzymatic biosensor that is capable of analyte sensing down to the low nanomolar concentration regime. This method is termed accumulation mode sensing and utilizes an immobilized redox polymer mediator wired to an oxidoreductase enzyme to store charge during a premeasurement charge concentration step, followed by a measurement step in which this accumulated charge is quantified. We demonstrate this new method using a model glucose sensor and show how the sensitivity of a sensor can be modified simply by adjusting the time duration of the charge concentration step. We achieve a limit of detection of 4.7 ± 1.4 nM using accumulation mode sensing, which represents a 25-fold improvement over traditional amperometry

PCR/ESI-MS primer and gene targets.

<p>PCR/ESI-MS primer and gene targets.</p

<i>B. burgdorferi</i> detections with untreated and IA treated whole blood extracts spiked with <i>B. burgdorferi</i> genomic DNA.

<p>Nucleic acid extracts from 1.25 mL of whole blood were spiked with decreasing of amounts of <i>B. burgdorferi</i> genomic DNA. In duplicate, specimens were either untreated or underwent IA before use in the PCR/ESI-MS <i>B. burgdorferi</i> detection/genotyping assay. The number of primer pairs in the assay detecting <i>B. burgdorferi</i> DNA was recorded for each specimen.</p

Comparison of isothermal amplification PCR/ESI-MS assay and two-tier serology for the detection of early Lyme disease on initial patient visit.

<p>Comparison of isothermal amplification PCR/ESI-MS assay and two-tier serology for the detection of early Lyme disease on initial patient visit.</p

Specimen results for IA/PCR/ESI-MS, initial, and follow-up two-tiered serology testing.

<p>Specimen results for IA/PCR/ESI-MS, initial, and follow-up two-tiered serology testing.</p

PCR/ESI-MS signatures of <i>Rickettsiaceae</i> and <i>Anaplasmataceae</i> species.

<p>PCR/ESI-MS signatures of <i>Rickettsiaceae</i> and <i>Anaplasmataceae</i> species.</p

Survey of <i>Ixodes pacificus</i> Ticks in California Reveals a Diversity of Microorganisms and a Novel and Widespread <i>Anaplasmataceae</i> Species

<div><p><i>Ixodes pacificus</i> ticks can harbor a wide range of human and animal pathogens. To survey the prevalence of tick-borne known and putative pathogens, we tested 982 individual adult and nymphal <i>I</i>. <i>pacificus</i> ticks collected throughout California between 2007 and 2009 using a broad-range PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to detect a wide range of tick-borne microorganisms. Overall, 1.4% of the ticks were found to be infected with <i>Borrelia burgdorferi</i>, 2.0% were infected with <i>Borrelia miyamotoi</i> and 0.3% were infected with <i>Anaplasma phagocytophilum</i>. In addition, 3.0% were infected with <i>Babesia odocoilei</i>. About 1.2% of the ticks were co-infected with more than one pathogen or putative pathogen. In addition, we identified a novel <i>Anaplasmataceae</i> species that we characterized by sequencing of its 16S rRNA, <i>groEL</i>, <i>gltA</i>, and <i>rpoB</i> genes. Sequence analysis indicated that this organism is phylogenetically distinct from known <i>Anaplasma</i> species with its closest genetic near neighbors coming from Asia. The prevalence of this novel <i>Anaplasmataceae</i> species was as high as 21% at one site, and it was detected in 4.9% of ticks tested statewide. Based upon this genetic characterization we propose that this organism be called ‘<i>Candidatus</i> Cryptoplasma californiense’. Knowledge of this novel microbe will provide awareness for the community about the breadth of the <i>I</i>. <i>pacificus</i> microbiome, the concept that this bacterium could be more widely spread; and an opportunity to explore whether this bacterium also contributes to human or animal disease burden.</p></div

Maximum-likelihood tree relationships among know <i>Anaplasmataceae</i> species and novel microorganism identified in this study inferred from (a) 16S rRNA, (b) <i>groEL</i>, (c) <i>gltA</i>, and (d) <i>rpoB</i> sequences.

<p>Sequences determined in this study are highlighted in bold. Numbers at nodes indicate percentages of bootstrap support based on 100 replicates. The horizontal bars correspond to substitutions per nucleotide position in panel (a) and to substitutions per amino acid position in panels (b-d).</p

Simpson's Diversity index (<i>DI</i>) of PCR/ESI-MS, rep-PCR and the combination of PCR/ESI-MS and rep-PCR.

<p>Simpson's Diversity index (<i>DI</i>) of PCR/ESI-MS, rep-PCR and the combination of PCR/ESI-MS and rep-PCR.</p

Predominant PCR/ESI-MS sequence types and rep-PCR types analyzed by Kullback-Leibler (KL) and Pearson correlation (PC) methods.

*<p>rep-PCR did not differentiate among ST 10, ST 12, ST 1 and ST 86.</p