Proteinase K Pretreatment for the Quantitative Recovery and Sensitive Detection of the Tuberculosis Biomarker Mannose-Capped Lipoarabinomannan Spiked into Human Serum.

This paper reports on an investigation of an enzymatic pretreatment protocol using proteinase K (ProK) for the analysis of human serum samples spiked with mannose-capped lipoarabinomannan (ManLAM). ManLAM is an antigenic biomarker found in the serum, urine, and other body fluids of individuals infected with tuberculosis (TB). Immunometric measurements of ManLAM are compromised by steric effects due to its complexation with high-molecular-weight components in these matrices that interfere with its capture and/or labeling.

Fluorescent sensor based on solid-phase extraction with negligible depletion: A proof-of-concept study with amines as analytes.

This paper describes the development and proof-of-concept testing of an easy-to-use trace analysis technique, namely F-SPE, by coupling fluorescent sensor with solid phase extraction (SPE). F-SPE is a two-step methodology that concentrates an analyte from a liquid sample onto a fluorophore-modified membrane and measures the amount of analyte from the extent the extracted analyte quenches the emission of the fluorophore. By applying the principle of negligible depletion (ND) intrinsic to SPE, the procedure of F-SPE for analyzing a sample can be markedly simplified while maintaining the ability to detect analytes at low limits of detection (LOD).

Molecularly-tunable nanoelectrode arrays created by harnessing intermolecular interactions.

Intermolecular interactions play a critical role in the binding strength of molecular assemblies on surfaces. The ability to harness them enables molecularly-tunable interfacial structures and properties. Herein we report the tuning of the intermolecular interactions in monolayer assemblies derived from organothiols of different structures for the creation of nanoelectrode arrays or ensembles with effective mass transport by a molecular-level perforation strategy.

Electrochemically Modulated Liquid Chromatography in Fused Silica Capillary Columns.

Electrochemically modulated liquid chromatography (EMLC) uses electrical potentials, applied to a conductive chromatographic stationary phase (e.g., porous graphitic carbon [PGC]), to manipulate analyte retention.

A simple vaporous probe with atomic-scale sensitivity to structural ordering and orientation of molecular assembly.

Understanding the structural ordering and orientation of interfacial molecular assemblies requires an insight into the penetration depth of the probe molecules which determines the interfacial reactivity. In contrast to the conventional liquid probe-based contact angle measurement in which penetration depth is complicated by the liquid cohesive interaction, we report here a new approach that features a simple combination of vaporous hexane, which involves only van der Waals interaction, and quartz crystal microbalance operated at the third harmonic resonance, which is sensitive to sub-monolayer (0.2%) adsorption.

SERS detection of neurotoxin serotypes A and B in buffer and serum: Towards the development of a biodefense test platform.

Botulinum neurotoxins (BoNTs) are classified at a highest degree of threat in biodefense, due largely to their high lethality. With the growing risk of biowarfare, the shortcomings of the gold standard test for these neurotoxins, the mouse bioassay, have underscored the need to develop alternative diagnostic testing strategies. This paper reports on the detection of inactivated neurotoxin serotype A (BoNT-A) and serotype B (BoNT-B), the two most important markers of botulism infection, by using a sandwich immunoassay, gold nanoparticle labels, and surface-enhanced Raman scattering (SERS) within the context of two threat scenarios.

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.

The Case for Human Serum as a Highly Preferable Sample Matrix for Detection of Anthrax Toxins.

This paper describes preliminary results on the surprising impact of human serum as a sample matrix on the detectability of protective antigen (PA) and lethal factor (LF), two antigenic protein markers of Bacillus anthracis, in a heterogeneous immunometric assay. Two sample matrices were examined: human serum and physiological buffer. Human serum is used as a specimen in the diagnostic testing of potentially infected individuals.

Investigation of Issues for the Accurate and Precise Measurement of an Analyte Using Surface-Enhanced Raman Scattering (SERS).

This paper builds on an earlier examination of the influence of sampling size and analyte surface density on the accuracy and precision of measurements using surface-enhanced Raman scattering (SERS) to read out heterogeneous immunoassays. Quantitation using SERS typically relies on interrogating a small area on the sample surface by using a micrometer-sized laser spot for signal generation. The information obtained using such a small portion of sample is then projected as being representative of the much larger sample, which can compromise the accuracy and precision of the measurement due to undersampling.

Detection of lipoarabinomannan in urine and serum of HIV-positive and HIV-negative TB suspects using an improved capture-enzyme linked immuno absorbent assay and gas chromatography/mass spectrometry.

TB diagnosis and treatment monitoring in resource limited regions rely heavily on serial sputum smear microscopy and bacterial culture. These microbiological methods are time-consuming, expensive and lack adequate sensitivity. The WHO states that improved TB diagnosis and treatment is imperative to achieve an end to the TB epidemic by 2030.

Gold Nanoparticle Labels and Heterogeneous Immunoassays: The Case for the Inverted Substrate.

This paper examines how the difference in the spatial orientation of the capture substrate influences the analytical sensitivity and limits of detection for immunoassays that use gold nanoparticle labels (AuNPs) and rely on diffusion in quiet solution in the antigen capture and labeling steps. Ideally, the accumulation of both reactants should follow a dependence governed by the rate in which diffusion delivers reactants to the capture surface. In other words, the accumulation of reactants should increase with the square root of the incubation time, i.

Adaptable Detection Strategies in Membrane-Based Immunoassays: Calibration-Free Quantitation with Surface-Enhanced Raman Scattering Readout.

This paper presents a method for immunometric biomarker quantitation that uses standard flow-through assay reagents and obviates the need for constructing a calibration curve. The approach relies on a nitrocellulose immunoassay substrate with multiple physical addresses for analyte capture, each modified with different amounts of an analyte-specific capture antibody. As such, each address generates a distinctly different readout signal that is proportional to the analyte concentration in the sample.

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).

Surface-enhanced Raman scattering II: concluding remarks.

Surface-enhanced Raman scattering (SERS) enables the detection of a large number of different adsorbates at extraordinarily low levels. This plasmonics-based technology has undergone a number of remarkable advances since its discovery over 40 years ago, and has emerged from being an investigative tool confined largely to the research laboratory into a much more usable tool across a broad range of investigative studies, both within the laboratory and beyond. The purpose of this Concluding remarks manuscript is to capture, at least in part, the developments in this area since the first Faraday discussion of SERS over a decade ago.

Colloidally Assembled Zinc Ferrite Magnetic Beads: Superparamagnetic Labels with High Magnetic Moments for MR Sensors.

Magnetic particles are widely used as labels in magnetoresistive sensors. To use magnetic particles as labels, several important characteristics should be considered, such as superparamagnetism, a high magnetic moment per particle (m), facile surface functionalization and biomolecule immobilization, colloidal stability, and analyte specificity. In this paper, we describe the preparation of magnetic labels with a high m, using colloidal assemblies of superparamagnetic zinc ferrite nanoparticles (ZFNPs, ∼9 nm).

Calibrant-Free Analyte Quantitation via a Variable Velocity Flow Cell.

In this paper, we describe a novel method for analyte quantitation that does not rely on calibrants, internal standards, or calibration curves but, rather, leverages the relationship between disparate and predictable surface-directed analyte flux to an array of sensing addresses and a measured resultant signal. To reduce this concept to practice, we fabricated two flow cells such that the mean linear fluid velocity, U, was varied systematically over an array of electrodes positioned along the flow axis. This resulted in a predictable variation of the address-directed flux of a redox analyte, ferrocenedimethanol (FDM).

Importance of specimen pretreatment for the low-level detection of mycobacterial lipoarabinomannan in human serum.

Patient care and prevention of disease outbreaks rely heavily on the performance of diagnostic tests. These tests are typically carried out in serum, urine, and other complex sample matrices, but are often plagued by a number of matrix effects such as nonspecific adsorption and complexation with circulating proteins. This paper demonstrates the importance of sample pretreatment to overcome matrix effects, enabling the low-level detection of a disease marker for tuberculosis (TB).

Detection of the tuberculosis antigenic marker mannose-capped lipoarabinomannan in pretreated serum by surface-enhanced Raman scattering.

The ability to detect tuberculosis (TB) continues to be a global health care priority. This paper describes the development and preliminary assessment of the clinical accuracy of a heterogeneous immunoassay that integrates a serum pretreatment process with readout by surface-enhanced Raman scattering (SERS) for the low-level detection of mannose-capped lipoarabinomannan (ManLAM). ManLAM is a major virulence factor in the infectious pathology of Mycobacterium tuberculosis (Mtb) that has been found in the serum and other body fluids of infected patients.

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.

Sampling Error: Impact on the Quantitative Analysis of Nanoparticle-Based Surface-Enhanced Raman Scattering Immunoassays.

This paper examines the impact of the sampling error caused by the small size of the focused laser spot when using surface-enhanced Raman scattering (SERS) as a quantitative readout tool to analyze a sandwich immunoassay. The assay consists of a thin-film gold substrate that is modified with a layer of capture monoclonal antibodies (mAbs) and extrinsic Raman labels (ERLs) that consist of gold nanoparticle cores (60 nm diameter) coated with a monolayer of a Raman reporter molecule and a layer of human IgG mAbs to tag the captured antigen. The contribution of sampling error to the measurement is delineated first by constructing and analyzing an antigenic random accumulation model; this is followed by an experimental study of the analysis of an assay substrate using two different laser spot sizes.

Prospects for point-of-care pathogen diagnostics using surface-enhanced Raman scattering (SERS).

Surface-enhanced Raman scattering (SERS) has enabled the detection of pathogens and disease markers at extremely low levels. This review examines the potential impact of SERS in addressing unmet needs in pathogen diagnostics both in a traditional clinical setting and in the point of care (POC) arena. It begins by describing the strengths and weaknesses of today's diagnostics technologies in order to set a contextual stage for an overview which highlights a few of the many recent developments using SERS in biodefense, human and animal health, and monitoring food and water safety.

Frequency-Domain Approach To Determine Magnetic Address-Sensor Separation Distance Using the Harmonic Ratio Method.

In this work, we describe an approach to determine the distance separating a magnetic address from a scanning magnetoresistive sensor, a critical adjustable parameter for certain bioassay analyses where magnetic nanoparticles are used as labels. Our approach is leveraged from the harmonic ratio method (HRM), a method used in the hard drive industry to control the distance separating a magnetoresistive read head from its data platter with nanometer resolution. At the heart of the HRM is an amplitude comparison of a signal's fundamental frequency to that of its harmonics.

Silica encapsulation of ferrimagnetic zinc ferrite nanocubes enabled by layer-by-layer polyelectrolyte deposition.

Stable suspensions of magnetic nanoparticles (MNPs) with large magnetic moment, m, per particle have tremendous utility in a wide range of biological applications. However, because of the strong magnetic coupling interactions often present in these systems, it is challenging to stabilize individual, high-moment, ferro- and ferrimagnetic nanoparticles. A novel approach to encapsulate large, that is, >100 nm, ferrimagnetic zinc ferrite nanocubes (ZFNCs) with silica after an intermediary layer-by-layer polyelectrolyte deposition step is described in this paper.

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.