In this work, the plasmonic gratings were integrated with detachable incubation wells through 24 well slide adapter. presence or absence of pulmonary tuberculosis while all individual urines were research ELISA LAM-negative. Plasmonic gratings produced by low-cost soft lithography were bound with anti-LAM capture antibody, Rabbit Polyclonal to ARRD1 incubated with patient urine samples, and biotinylated detection antibody. Fluorescently labeled streptavidin revealed single molecule emission by epifluorescence microscope. Using a 1 fg/mL baseline for limit of detection, single molecule FLISA exhibited good qualitative agreement with gold standard assessments on 19 of 20 patients, including trans-Zeatin accurately predicting the gold-standard-negative patients, trans-Zeatin while one gold-standard-positive patient produced no observable LAM in urine. Conclusions Single molecule FLISA by plasmonic grating exhibited the ability to quantify tuberculosis LAM from complex urine samples of patients from a high endemic setting with negligible interference from your complex media itself. Moreover, agreement with patient diagnoses by platinum standard testing suggests that single molecule FLISA could be used as a highly sensitive test to diagnose tuberculosis noninvasively. Introduction Estimates vary, but recent work suggests that greater than one quarter of incident tuberculosis (TB) cases went undiagnosed or unreported in 2018 . Timely access to diagnosis is critical for disease management; however, several disease-intrinsic and engineering challenges compromise the development of quick, near-patient, diagnostic assessments. Sputum is usually a widely-used input for diagnosis, but the complexity and heterogeneity of this sample matrix require specialized gear and/or infrastructure, thus, limiting time-to-detection (TTD) and point-of-care (POC) deployment. A recent example is the rollout of the PCR-based GeneXpert MTB/RIF test, which incorporates a largely automated workflow from patient sputum sample to result and TTD is only a few hours. However, GeneXpert is relatively expensive ($10 per test after hardware) and requires operator training, instrument upkeep, and calibration . Furthermore, not all patients have TB disease that leads trans-Zeatin to sputum production, including children and HIV positive individuals, making timely diagnosis of these populations particularly challenging [3C5]. Reducing reliance on sputum, utilizing alternative sample sources, considering option TB and TB disease biomarkers, assay simplification, and decreasing cost are all important parameters that will lead to novel diagnostics that inform an affordable POC test. Lipoarabinomannan (LAM) is usually a prominent glycolipid of the mycobacterial cell wall that has received substantial attention as a TB disease-associated biomarker [6C8]. LAM can be detected from sputum, blood, and urine samples, and can be detected in routine immunologic tests such as enzyme-linked immunosorbent or lateral circulation assays (ELISA or LFA, respectively) [9, 10]. To this, the Alere Determine TB LAM Ag Test [Abbott Diagnostics, Santa Clara, CA, USA] is usually a commercially available quick LFA LAM diagnostic test. While demonstrating the feasibility of LAM as a TB biomarker, the sensitivity/specificity profile led the World Health Business (WHO) to limit its endorsement to a subgroup of HIV+ adult in-patients with advanced disease and CD4+ T-cell counts 100 cell/L and signs and symptoms of active TB disease . Other recently published studies on LAM detection assays report relatively high limits of detection (LOD), between 10C0.05 ng/mL, which still may not enable reliable, routine TB diagnosis regardless of HIV and CD4 status [12, 13]. Increasing sensitivity/lowering the LOD is usually a encouraging avenue for assessing whether or not LAM could serve as a more broadly useful TB biomarker. One approach generating a measure of success at improving LODs and clinical performance is to capture LAM from larger volumes [14, 15]. Others statement an electrochemiluminescence-based ELISA format to achieve LODs in a similar range, 350C650 fM (6C11pg/mL), and exhibited the ability for the assay to successfully detect LAM in patient urines . While this is 3 logs more sensitive than standard LFA/ELISA methods, we hypothesized that additional increases in sensitivity by enhancing signal-to-noise ratio (SNR) would permit the assessment of LAM as a TB diagnostic biomarker from clinically relevant samples. In the present work, we describe a fluorescence-enhancing plasmonic grating platform combined with a fluorescence-linked immunosorbent assay (FLISA) for LAM detection.
In this work, the plasmonic gratings were integrated with detachable incubation wells through 24 well slide adapter