Detection of the tuberculosis biomarker mannose-capped lipoarabinomannan in human serum: Impact of sample pretreatment with perchloric acid.

The development of an accurate and rapid diagnostic test for tuberculosis (TB) to use at point of need is vital to efforts aimed at reducing the global burden from this disease. This paper builds on our previous studies of mannose-capped lipoarabinomannan (ManLAM) as a serum biomarker for active TB infection by means of a heterogeneous immunoassay. That work found that complexation with components in serum (e.g., proteins) sterically hindered the capture and/or labeling of ManLAM in an immunoassay at levels <10 ng mL-1, compromising the clinical utility of this biomarker for detection of active TB infection. We also showed that the acidification of ManLAM-containing serum samples with perchloric acid improved the detectability of ManLAM by 250× by complex disruption when compared to measurements of untreated serum. The present study examined what effects the PCA treatment of serum samples may have on the recovery and structural integrity of ManLAM, owing to its potential susceptibility to acid hydrolysis. Recovery was assessed with an enzyme-linked immunosorbent assay (ELISA). The possible impact of acid hydrolysis on the ManLAM structure was investigated by gas chromatography-mass spectrometry and carbohydrate chemical degradation methods. The ELISA study indicated that while the signal strength for ManLAM in the serum spike-in experiments was significantly stronger after PCA pretreatment when compared to untreated human serum, it was only ∼20% of the ManLAM measured in physiological buffer. This loss in detectability was shown by structural analysis to arise mainly from the acid-induced degradation of the arabinan domains of ManLAM that are targeted by antibodies used for antigen capture and/or tagging. The implications of these findings in terms of the detection of this important biomarker for TB are also discussed.

Handheld Raman Spectrometer Instrumentation for Quantitative Tuberculosis Biomarker Detection: A Performance Assessment for Point-of-Need Infectious Disease Diagnostics.

Techniques for the detection of disease biomarkers are key components in the protection of human health. While work over the last few decades has redefined the low-level measurement of disease biomarkers, the translation of these capabilities from the formal clinical setting to point-of-need (PON) usage has been much more limited. This paper presents the results of experiments designed to examine the potential utility of a handheld Raman spectrometer as a PON electronic reader for a sandwich immunoassay based on surface-enhanced Raman scattering (SERS). In so doing, the study herein used a recently developed procedure for the SERS detection of phospho-myo-inositol-capped lipoarabinomannan (PILAM) as a means to compare the performance of laboratory-grade and handheld instrumentation and, therefore, gauge the utility of the handheld instrument for PON deployment. Phospho-myo-inositol-capped lipoarabinomannan is a non-pathogenic simulant for mannose-capped lipoarabinomannan (ManLAM), which is an antigenic marker found in serum and other body fluids of individuals infected with tuberculosis (TB). The results of the measurements with the field-portable spectrometer were then compared to those obtained for the same samples when using a much more sensitive benchtop Raman spectrometer. The results, albeit under different operational settings for the two spectrometers (e.g., signal integration time), are promising in that the limit of detection found for PILAM spiked in human serum when using the handheld system (0.18 ng/mL) approached that of the benchtop instrument (0.032 ng/mL). This work also: (1) identified potential adaptations (e.g., optimization of the plasmonically enhanced response for measurement by the handheld unit through a change in the excitation wavelength) to tighten the gap in performance; and (2) briefly examined the next steps and potential processes required to move this immunoassay platform closer to PON utility.

Advantages and limitations of nanoparticle labeling for early diagnosis of infection.

INTRODUCTION: Nanoparticle-based disease diagnostics harness a range of unique physical and chemical phenomena for the detection of biomarkers at exceedingly low levels. This capability potentially enables the diagnosis of disease earlier in its progression and improves the likelihood of positive treatment outcomes. This review highlights recent work in this area, and then projects the next steps needed to move this emerging capability beyond the research laboratory. AREAS COVERED: This review examines the advantages and limitations of in vitro health care diagnostic tests that utilize nanoparticles (e.g. noble metal, quantum dot, and magnetic). It includes a brief overview of their unique properties, syntheses, and applicable readout strategies. This is followed by a brief synopsis of the obstacles faced when attempting to translate nanoparticle-based diagnostics from the R&D laboratory to the clinic and other arenas (i.e. the difficulties common to in vitro diagnostics), and then by a much more in-depth examination of the need to control and characterize a range of nanoparticle properties (e.g. size, shape, surface composition, and stability) when making this transition. Expert commentary: The review wraps up with a short commentary and perspective for the next five years, focusing on possible guidelines for nanoparticle characterization.

Succinimidyl ester surface chemistry: implications of the competition between aminolysis and hydrolysis on covalent protein immobilization.

N-Hydroxysuccinimide (NHS) ester terminal groups are commonly used to covalently couple amine-containing biomolecules (e.g., proteins and peptides) to surfaces via amide linkages. This one-step aminolysis is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry. The work herein examines the efficiency of covalent protein immobilization in borate buffer (50 mM, pH 8.50) using the thiolate monolayer formed by the chemisorption of dithiobis (succinimidyl propionate) (DSP) on gold films. The structure and reactivity of these adlayers are assessed via infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), electrochemical reductive desorption, and contact angle measurements. The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of the interfacial reaction on the packing and presence of defects in the adlayer. This interpretation is used in the subsequent analysis of IR-ERS kinetic plots which give a heterogeneous aminolysis rate constant, ka, that is over 3 orders of magnitude lower than that of the heterogeneous hydrolysis rate constant, kh. More importantly, a projection of these heterogeneous kinetic rates to protein immobilization suggests that under coupling conditions in which low protein concentrations and buffers of near physiological pH are used, proteins are more likely physically adsorbed rather than covalently linked. This result is paramount for biosensors that use NHS chemistry for protein immobilization due to effects that may arise from noncovalently linked proteins.