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. Recent work has shown that deproteinization of these types of samples by perchloric acid acidification or ProK digestion releases ManLAM from complexation. Releasing ManLAM greatly improves its detectability and, as a result, its utility as a TB biomarker. The work detailed herein examined how different ProK reaction conditions (e.g., enzyme concentration and digestion time and temperature) affect the recovery and detectability of ManLAM in human serum. As measured by enzyme-linked immunosorbent assay (ELISA), we show that using the optimal set of digestion conditions to free ManLAM, which also yield a small, quantitatively reproducible level of sample concentration, it is possible to achieve a spiked ManLAM recovery of 98 ± 13% and a limit of detection of 10 pg/mL (0.6 pM). Experiments also demonstrated that the ELISA responses measured for a given ManLAM concentration in serum after pretreatment were statistically indistinguishable from those directly determined for the same amounts of ManLAM added to an innocuous buffered solution. Possible adaptations of the digestion protocol for use in point-of-care TB testing are also briefly discussed.

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). The merits of this approach are demonstrated by impregnating a SPE membrane with a perylene diimide (PDI) fluorophore, N,N'-di(nonyldecyl)-perylene-3,4,9,10-tetracarboxylic diimide (C9/9-PDI), for the low-level detection of organic amines (e.g., aniline) and amine-containing drugs (e.g., Kanamycin). The sensing mechanism is based on the donor-acceptor quenching of PDI by amines, which, when coupled with the concentrative nature of SPE, yields LODs for aniline and Kanamycin of 67 nM (∼6 ppb) and 32 nM (∼16 ppb), respectively.

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. The homo- and hetero-intermolecular interactions can be fully controlled, which is demonstrated not only by thermodynamic analysis of the fractional coverage but also by surface infrared reflection absorption and X-ray photoelectron spectroscopic characterizations. This understanding enables controllable electrochemical perforation for the creation of ensembles or arrays of channels across the monolayer thickness with molecular and nanoscale dimensions. Redox reactions on the nanoelectrode array display molecular tunability with a radial diffusion characteristic in good agreement with theoretical simulation results. These findings have implications for designing membrane-type ion-gating, electrochemical sensing, and electrochemical energy storage devices with molecular level tunability.

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. This paper reports the design of a capillary EMLC column with a smaller internal diameter (ID; 250 µm) than that of the standard bore predecessor (3.3 mm ID). The new capillary EMLC columns are configured so that the PGC stationary phase serves as the working electrode in a two-electrode electrochemical cell and simplifies electrode placement by obviating the need for a counter electrode. This configuration also eliminates the internal Nafion sleeve that is critical to operation for the standard bore columns, thereby avoiding Nafion deformation as a source of chromatographic band broadening and rupturing as a mode of column failure. Indeed, values for chromatographic efficiency obtained on the capillary columns meet or exceed those measured for the standard columns (20 000-40 000 vs 14 000 plates/m, respectively) with near symmetric elution bands (asymmetry factors of 1.1-1.4 for well-packed capillaries) that surpass band symmetries observed in all prior studies. A test suite of aromatic sulfonates was used to characterize the chromatographic performance of the capillary EMLC columns. Separations of this test mixture showed that retention factors for individual analytes could be manipulated by as much as 21× by changing the applied potential at the PGC stationary phase. Changes in retention behavior at different potential ranges, hypothesized to result from differences in adsorption orientation, were also observed and are consistent with past work. Collectively, the retention behavior unique to EMLC is operative in this new capillary configuration and promises to open new avenues in tuning LC separations.

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. Using this combination, we demonstrated the ability of probing the structural ordering and orientation of the self-assembled monolayers with a sensitivity from penetrating the top portion of the monolayers to interacting with the very top atomic structure at the interface. The determination of the dependence of the adsorption energy of vaporous hexane on the penetration depth in the molecular assembly allowed us to further reveal the atomic-scale origin of the odd-even oscillation, which is also substantiated by density functional theory calculations. The findings have broader implications for designing interfacial reactivities of molecular assemblies with atomic-scale depth precision.

SERS detection of Clostridium botulinum 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 Clostridium botulinum 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. The first scenario mimics part of the analysis needed in response to a "white powder" threat by measuring both neurotoxins in phosphate-buffered saline (PBS), a biocompatible solvent often used to recover markers dispersed in a powdered matrix. The second scenario detects the two neurotoxins in spiked human serum to assess the clinical potential of the platform. The overall goal is to develop a test applicable to both scenarios in terms of projections of required levels of detection. We demonstrate the ability to measure BoNT-A and BoNT-B in PBS at a limit of detection (LoD) of 700 pg/mL (5 pM) and 84 pg/mL (0.6 pM), respectively, and in human serum at 1200 pg/mL (8 pM) and 91 pg/mL (0.6 pM), respectively, with a time to result under 24 h. The steps required to transform this platform into an onsite biodefense screening tool that can simultaneously and rapidly detect (<1 h) these and other agents are briefly discussed.

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.

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. For a heterogeneous immunoassay interrogated by SERS, quantitation is, therefore, sensitive to the size of the analyzed area and the surface density of the measured analyte. To identify conditions in which sampling error poses a threat to accuracy and precision, a simulation of a SERS immunoassay was developed and compared to experimental results. The simulation randomly distributes adsorbates across the capture surface and then measures the density of adsorbates inside areas of analysis of different sizes. This approach mimics the analysis of a heterogeneous immunoassay when using a Raman microscope with different laser spot sizes. The results of the simulations, which were confirmed experimentally by comparison to an immunoassay of human immunoglobulin G (IgG) show that the accuracy and precision of the measurement improved with larger analysis areas and higher analyte concentrations due to the increased apparent homogeneity of the analyte within the area of analysis. By imposing a threshold on precision (5%), we also begin to establish a framework for the parameters necessary to achieve reliable quantitative measurements (e.g., laser spot size, analyte concentration, and sample volume).

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. Physiological buffers are often applied to the recovery of biomarkers dispersed in suspicious white powders and other suspect specimens and as a serum diluent to combat contributions to the measured test response from nonspecific adsorption. The results of these experiments using a sandwich immunoassay read out by surface-enhanced Raman scattering yielded estimates for the limit of detection (LOD) for both markers when using spiked human serum that were remarkably lower than those of spiked physiological buffer (∼70,000× for PA and ∼25,000× for LF). The difference in LODs is attributed to a degradation in the effectiveness of the capture and/or labeling steps in the immunoassay due to the known propensity for both proteins to denature in buffer. These findings indicate that the use of physiological buffer for serum dilution or recovery from a powdered matrix is counter to the low-level detection of these two antigenic proteins. The potential implications of these results with respect to the ability to detect markers of other pathogenic agents are briefly discussed.

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. Commercially available lipoarabinomannan (LAM) detection tools perform at low sensitivity that are highly dependent on the underlying immunological status of the patient; those with advanced HIV infection perform well. In this study, we have applied two novel strategies towards the sensitive diagnosis of TB infection based on LAM: Capture ELISA to detect LAM in paired urine and serum samples using murine and human monoclonal antibodies, essentially relying on LAM as an 'immuno-marker'; and, secondly, detection of α-d-arabinofuranose and tuberculostearic acid (TBSA)- 'chemical-markers' unique to mycobacterial cell wall polysaccharides/lipoglycans by our recently developed gas chromatography/mass spectrometry (GC/MS) method. Blinded urine specimens, with microbiologically confirmed active pulmonary TB or non TB (HIV+/HIV-) were tested by the aforementioned assays. LAM in patient urine was detected in a concentration range of 3-28 ng/mL based on GC/MS detection of the two LAM-surrogates, d-arabinose and tuberculostearic acid (TBSA) correctly classifying TB status with sensitivity > 99% and specificity = 84%. The ELISA assay had high sensitivity (98%) and specificity (92%) and the results were in agreement with GC/MS analysis. Both tests performed well in their present form particularly for HIV-negative/TB-positive urine samples. Among the HIV+/TB+ samples, 52% were found to have >10 ng/mL urinary LAM. The detected amounts of LAM present in the urine samples also appears to be associated with the gradation of the sputum smear, linking elevated LAM levels with higher mycobacterial burden (odds ratio = 1.08-1.43; p = 0.002). In this small set, ELISA was also applied to parallel serum samples confirming that serum could be an additional reservoir for developing a LAM-based immunoassay for diagnosis of TB.

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.e., t1/2. The work herein shows, however, that this expectation is only obeyed when the capture substrate is oriented to direct the gravity-induced sedimentation of the AuNP labels away from the substrate. Using an assay for human IgG, the results show that circumventing the sedimentation of the gold nanoparticle labels by substrate inversion enables the dependence of the labeling step on diffusion, reduces nonspecific label adsorption, and improves the estimated detection limit by ∼30×. High-density maps of the signal across the two types of substrates also demonstrate that inversion in the labeling step results in a more uniform distribution of AuNP labels across the surface, which translates to a greater measurement reproducibility. These results, which are supported by model simulations via the Mason-Weaver sedimentation-diffusion equation, and their potential implications when using other nanoparticle labels and related materials in diagnostic tests and other applications, are briefly discussed.

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. To establish the feasibility of this concept, equations derived from antibody-antigen binding equilibrium were first applied in modeling experiments. Next, nitrocellulose membranes with multiple capture antibody addresses were fabricated for detection of a model analyte, human Immunoglobulin G (hIgG), by a heterogeneous sandwich immunoassay using antibody-modified gold nanoparticles (AuNPs) as the immunolabel. Counting the number of colored capture addresses visible to the unassisted eye enabled semiquantitative hIgG determination. We then demonstrated that, by leveraging the localized surface plasmon resonance of the AuNPs, surface-enhanced Raman spectroscopy (SERS) can be used for quantitative readout. By comparing the SERS signal intensities from each capture address with values predicted using immunoassay equilibrium theory, the concentration of hIgG can be determined (∼30% average absolute deviation) without reference to a calibration curve. This work also demonstrates the ability to manipulate the dynamic range of the assay over ∼4 orders of magnitude (from 2 ng mL-1 to 10 µg mL-1). The potential prospects in applying this concept to point-of-need diagnostics 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.

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. It begins with a brief contextual overview and then moves into describing a few of the many highlights from the meeting. Along the way, we have added a few comments and perspectives as a means to more fully stage where the different areas of research with SERS stand today. An addendum is included that collects a few of the recent perspectives on the original work and activities in this area.

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). Also, several properties of these particles are compared with those of commercially available magnetic beads, Dynabeads and TurboBeads. The colloidally assembled zinc ferrite magnetic beads (ZFMBs, ∼160 nm) were synthesized by assembling ZFNPs via an emulsion-based assembly approach. While retaining superparamagnetism at room temperature, the m of ZFMBs is ∼4000× higher than that of the constituent ZFNPs. Surface functionalization with a layer of polyacrylic acid stabilized the ZFMBs in aqueous solution and enabled conjugation with streptavidin via carbodiimide linking chemistry. The streptavidinated ZFMBs can be suspended in aqueous buffer for ≥24 h, whereas 1.05 µm Dynabeads and 30 nm TurboBeads undergo ballistic deposition and instantaneous aggregation in solution, respectively. Finally, the streptavidinated ZFMBs were employed as labels in an immunoassay for the detection of osteopontin, a potential pancreatic cancer marker, proving superior to the commercial particles in terms of limit of detection and dynamic range. We expect that the work presented in this article can be extended to other biological applications, especially where superparamagnetic particles with a high m and colloidal stability are needed.

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). The resultant limiting currents measured at a series of these electrodes, and accurately described by a convective-diffusive transport model, provided a means to calculate an "unknown" concentration without the use of calibrants, internal standards, or a calibration curve. Furthermore, the experiment and concentration calculation only takes minutes to perform. Deviation in calculated FDM concentrations from true values was minimized to less than 0.5% when empirically derived values of U were employed.

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). The impact of pretreatment is illustrated by detecting a cell wall component unique to mycobacteria, lipoarabinomannan (LAM). LAM is a major virulence factor in the infectious pathology of Mycobacterium tuberculosis (Mtb) and has been successfully detected in the body fluids of TB-infected individuals; however, its clinical sensitivity - identifying patients with active infection - remains problematic. This and the companion paper show that the detection of LAM in an immunoassay is plagued by its complexation with proteins and other components in serum. Herein, we present the procedures and results from an investigation of several different pretreatment schemes designed to disrupt complexation and thereby improve detection. These sample pretreatment studies, aimed at determining the optimal conditions for complex disruption, were carried out by using a LAM simulant derived from the nonpathogenic M. smegmatis, a mycobacterium often used as a model for Mtb. We have found that a perchloric acid-based pretreatment step improves the ability to detect this simulant by ∼1500× with respect to that in untreated serum. This paper describes the approach to pretreatment, how pretreatment improves the detection of the LAM simulant in human serum, and the results from a preliminary investigation to identify possible contributors to complexation by fractionating serum according to molecular weight. The companion paper applies this pretreatment approach to assays of TB patient samples.

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. The effectiveness of ManLAM as a TB diagnostic marker, however, remains unproven for reasons not yet well understood. As reported herein, we have found that (1) ManLAM complexes with proteins and possibly other components in serum; (2) these complexes have a strongly detrimental impact on the ability to detect ManLAM using an immunoassay; (3) a simple pretreatment step can disrupt this complexation; and (4) disruption by pretreatment improves detection by 250×. We also describe the results from a preliminary assessment on the utility of serum pretreatment by running immunoassays on archived specimens from 24 TB-positive patients and 10 healthy controls. ManLAM was measurable in 21 of the 24 TB-positive specimens, but not in any of the 10 control specimens. These findings, albeit for a very small specimen set, translate to a clinical sensitivity of 87.5% and a clinical specificity of 100%. Together, these results both provide much needed evidence for the clinical utility of ManLAM as a TB marker, and demonstrate the potential utility of our overall approach to serve as a new strategy for the development of diagnostic tests for this disease.

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.

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. Both sets of findings indicate that the analysis with a small laser spot can lead to a sampling error (i.e., undersampling) much like that found when the size of a measured soil sample fails to accurately match that of a larger, more representative sample. That is, the smaller the laser spot size, the larger probable deviation in the accuracy of the measurement and the greater the imprecision of the measurement. Possible implications of these results with respect to the general application of SERS for quantitative measurements are also briefly discussed.