TOWARDS HOME MONITORING OF ANTICONVULSANT LEVELS: A PILOT STUDY USING BIO-NANO-CHIP TECHNOLOGY
Authors: G. Kalamangalam, N. Christodoulides, N. Ali, J. Hohenstein, R. Farid, J. Lindo-Gibbs, A. Dasgupta, J. Wentworth, E. Lykissa, J. McDevitt
With >15 marketed antiepileptic drugs (AEDs), pharmacotherapy remains the mainstay of epilepsy treatment. Serial serum AED measurements can be crucial in managing a patient’s dosing; however, it is impractical for serum levels to be monitored too frequently. There is an urgent need for alternative AED assay technologies that are non-invasive, repeatable, adapted to domiciliary use, and cost-effective. Bio-nano-chips (BNCs) are a new generation of compact programmable chemical processors that may be used to assay a variety of biological analytes (Jokerst & McDevitt, Nanomedicine, 5(1), 143-55, 2010). Here we report progress towards developing a prototype BNC device – a ‘lab-on-a-chip’ - for measuring AED levels from saliva that addresses the above needs.
(i) BNC calibration: Multiplexed BNC-based competitive immunoassays for two commonly used AEDs (analytes) – phenytoin (PHT) and phenobarbital (PHB) – were developed on bead-based technology. Drug-specific sensitized beads were blocked with 1% bovine serum albumin/phosphate buffered saline (BSA/PBS), and exposed to AlexaFluor-488®-conjugated tracer in the presence of assay vehicle alone or with drug (standard or sample) to obtain baseline optical signals and a ‘competitive mix’, respectively. Assays were optimized by experimenting with multiple assay parameters. The limit of detection (LOD) and useful range of the assay were computed. (ii) Patient specimens: Epilepsy patient volunteers on one or both of chronic PHT/PHB therapy were recruited in an outpatient setting. At each encounter, patients provided a single serum sample and multiple salivary samples. Saliva was either collected by passive drool (‘clear saliva’; CS) or was obtained by oral swab and diluted into an Aware Messenger® transfer matrix (‘buffered saliva’; BS) and frozen at -80C for later analysis. (iii) ‘Gold standard’ testing: Serum samples were processed by particle enhanced turbidimetric immunoassay (PETINIA) in a clinical chemistry laboratory. A control group of salivary samples were suspended in PBS and assayed by gas chromatography-mass spectrometry (GC-MS) at a commercial toxicology laboratory. iv) BNC testing of patient samples: CS and BS samples were further quantitatively diluted in Aware Messenger® and assays were run using a sequentially diluted competitive mix against 3 clinical samples of PHT and 5 of PHB (total n=8).
BNC calibration signals were robust and provided low, reliable limits of detection (Figures 1a-b). BNC results of clinical samples for both PHT and PHB compared favorably with those obtained by GC-MS (Table 1: shaded regions highlight pairs of gold-standard and BNC readings for the same sample type).
We provide proof-of-concept for a realistic saliva-based BNC assay system for two commonly used AEDs, validating our results against gold-standards. Further work aims to produce a practical point-of-care diagnostic of this type – eventually a handheld device the size of a credit card - for multiple AEDs that will empower patients to monitor their own drug levels in the community.
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