Detection of small molecules with a flow immunosensor

We describe the development of an easy-to-use sensor with widespread applications for detecting small molecules. The flow immunosensor can analyze discrete samples in under one minute or continuously monitor a flowing stream for the presence of specific analytes. This detection system is extremely specific, and achieves a level of sensitivity which meets or exceeds the detection limits reported for rival assays. Because the system is also compact, transportable, and automated, it has the potential to impact diverse areas. For example, the flow immunosensor has successfully detected drugs of abuse and explosives, and may well address many of the needs of the environmental community with respect to continuous monitoring for pollutants. Efforts are underway to engineer a portable device in the field

Fluorescence-based Sensing of 2,4,6-Trinitrotoluene (TNT) Using a Multi-channeled Poly(methyl methacrylate) (PMMA) Microimmunosensor

Fluorescence immunoassays employing monoclonal antibodies directed against the explosive 2,4,6-trinitrotoluene (TNT) were conducted in a multi-channel microimmunosensor. The multi-channel microimmunosensor was prepared in poly (methyl methacrylate) (PMMA) via hot embossing from a brass molding tool. The multi-channeled microfluidic device was sol-gel coated to generate a siloxane surface that provided a scaffold for antibody immobilization. AlexaFluor-cadaverine-trinitrobenzene (AlexaFluor-Cad-TNB) was used as the reporter molecule in a displacement immunoassay. The limit of detection was 1–10 ng/mL (ppb) with a linear dynamic range that covered three orders of magnitude. In addition, antibody crossreactivity was investigated using hexahydro-1,3,5-triazine (RDX), HMX, 2,4-dinitrotoluene (DNT), 4-nitrotoluene (4-NT) and 2-amino-4,6-DNT

Application of Broad-Spectrum, Sequence-Based Pathogen Identification in an Urban Population

A broad spectrum detection platform that provides sequence level resolution of target regions would have a significant impact in public health, case management, and means of expanding our understanding of the etiology of diseases. A previously developed respiratory pathogen microarray (RPM v.1) demonstrated the capability of this platform for this purpose. This newly developed RPM v.1 was used to analyze 424 well-characterized nasal wash specimens from patients presenting with febrile respiratory illness in the Washington, D. C. metropolitan region. For each specimen, the RPM v.1 results were compared against composite reference assay (viral and bacterial culture and, where appropriate, RT-PCR/PCR) results. Across this panel, the RPM assay showed ≥98% overall agreement for all the organisms detected compared with reference methods. Additionally, the RPM v.1 results provide sequence information which allowed phylogenetic classification of circulating influenza A viruses in ∼250 clinical specimens, and allowed monitoring the genetic variation as well as antigenic variability prediction. Multiple pathogens (2–4) were detected in 58 specimens (13.7%) with notably increased abundances of respiratory colonizers (esp. S. pneumoniae) during viral infection. This first-ever comparison of a broad-spectrum viral and bacterial identification technology of this type against a large battery of conventional “gold standard” assays confirms the utility of the approach for both medical surveillance and investigations of complex etiologies of illness caused by respiratory co-infections

CONTINUOUS FLOW IMMUNOASSAY: USE OF A NOVEL TRIFUNCTIONAL CARRIER MOLECULE FOR THE SYNTHESIS OF FLUOROPHORE-LABELED ANTIGENS

We developed a fluorescent immunosensor operating in continuous flow and capable of detecting low molecular weight antigens. The approach differs from previously described continuous flow assays by not requiring incubation steps or the introduction of reagents following the loading of the sample into the system. Detection of the antigen is rapid, occurring within three minutes in the system described. The assay is based on the binding of labeled antigen to an immobilized antibody, with subsequent displacement of the labeled antigen when antigen is present in the buffer flow. In order to increase the sensitivity of the assay, we developed a novel trifunctional carrier molecule for the fluorescent labeling of the antigen. The backbone of the carrier consists of the 21 amino acid residues of the insulin A-chain, which provides a single site (terminal amino group) for covalent coupling of the antigen, three carboxyl groups for the attachment of fluorophores, and four sulfhydryl groups for derivatization with hydrophilic residues to compensate for the hydrophobic effect of the fluorophores. In this study, the model antigen 2,4-dinitrophenol (DNP) was coupled to the terminal amino group, the sulfhydryl groups were oxidized to S-sulfonates, and the carboxyl groups were derivatized with fluorescein using carbohydrazide as spacer. The properties of the DNP-insulin A-chainfluorescein conjugate (DNP-Ins-Fl) were compared to those of a DNP derivative labeled with a single fluorescein residue via a small lysine spacer (DNP-Lys-Fl). At equimolar concentrations the DNP-Ins-Fl generated a 2.6-fold higher fluorescent signal than the DNP-Lys-Fl, and exhibited a three-fold lower nonspecific adsorption to immobilized nonimmune IgG. Due to these properties of DNP-Ins-Fl,as little as 50 pmol of DNP-lysine could be detected in the fluorescent continuous flow immunoassay

Method Development for Metaproteomic Analyses of Marine Biofilms

The large-scale identification and quantitation of proteins via nanoliquid chromatography (LC)-tandem mass spectrometry (MS/MS) offers a unique opportunity to gain unprecedented insight into the microbial composition and biomolecular activity of true environmental samples. However, in order to realize this potential for marine biofilms, new methods of protein extraction must be developed as many compounds naturally present in biofilms are known to interfere with common proteomic manipulations and LC-MS/MS techniques. In this study, we used amino acid analyses (AAA) and LC-MS/MS to compare the efficacy of three sample preparation methods [6 M guanidine hydrochloride (GuHCl) protein extraction + in-solution digestion + 2D LC; sodium dodecyl sulfate (SDS) protein extraction + 1D gel LC; phenol protein extraction + 1D gel LC] for the metaproteomic analyses of an environmental marine biofilm. The AAA demonstrated that proteins constitute 1.24% of the biofilm wet weight and that the compared methods varied in their protein extraction efficiencies (0.85–15.15%). Subsequent LC-MS/MS analyses revealed that the GuHCl method resulted in the greatest number of proteins identified by one or more peptides whereas the phenol method provided the greatest sequence coverage of identified proteins. As expected, metagenomic sequencing of the same biofilm sample enabled the creation of a searchable database that increased the number of protein identifications by 48.7% (≥1 peptide) or 54.7% (≥2 peptides) when compared to SwissProt database identifications. Taken together, our results provide methods and evidence-based recommendations to consider for qualitative or quantitative biofilm metaproteome experimental design