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This study describes a novel technique for direct analysis of thiocyanate in human saliva using negative electrospray. ion mobility spectrometry (ESI-IMS) and without any considerable sample pretreatment. The ESI-IMS system with nearly complete desolvation cannot be useful for salivary thiocyanate analysis because complete overlapping occurs between the format peak of the solvent and the thiocyanate peak. In addition, the chloride ions existing in saliva produce a very broad peak, which has a drastically interfering effect on [SCN.sup.-] determination. In this study, with a little change in the operation conditions of the apparatus (lowering the flow rate of the drift gas), it was possible to overcome these problems. The desolvation process was decreased in the lower flow of drift gas, and this caused the [SCN.sup.-] peak to be separated completely from other interference peaks such as chloride ions. The results obtained in this proposed methodology provide the detection limit of 0.003 [micro]g/mL and the linear dynamic range from 0.01 to 1.00 [micro]g/mL for the thiocyanate. Several human saliva samples were analyzed with the proposed negative ESI-IMS, and the satisfactory results confirm the capability of the method for direct analysis of salivary thiocyanate. 10.1021/ac101017m

In the present study, the ability of [sup.1]H nuclear magnetic resonance (NMR) for metabolic profiling of human saliva samples was investigated. High-resolution ]H NMR spectra were obtained, and signals were assigned to various metabolites mainly representing small organic acids and amino acids. In addison, the use of human saliva for metabolomic studies was evaluated, and multivariate data analysis revealed that the 92 morning and night samples from 46 subjects could be discriminated with a predictability of 85%. The diurnal effect on the salivary metabolite profile were ascribed to changes in intensities of several metabolites including trimethylamine oxide (TMAO), choline, propionate, alanine, methanol, and N-acetyl groups. No effects of gender and body mass index (BMI) on the salivary metabolite profile were detected. The relationships between the salivary metabolome and glycated hemoglobin, systolic and diastolic blood pressure were investigated; however, no significant correlations could be established. 10.1021/ac9020598

This current study presents a new miniature, integrated system capable of rapid detection of genetic deletion from saliva samples. Several critical modules including a genomic DNA (gDNA) extraction module, a polymerase chain reaction (PCR) module, and an external optical detection module are integrated into the system. Silica-modified magnetic beads are first incubated with saliva in an extraction chamber with a cell lysis solution. This is followed by the collection of released gDNA onto the surface of the microbeads, which is then further purified and concentrated utilizing a magnetic field generated by an on-chip array of microcoils. Then, genetic deletion of human genes can be specifically amplified by the on-chip PCR module and is immediately detected using the optical detection module. Experimental results show that high-quality gDNAwith an average concentration of 50.45 ng/ [micro]L can be extracted from 100 [micro]L of saliva. The detection of a mutated [alpha]-globin gene associated with [alpha]-thalassemia-1 of southeast Asian (SEA)-type deletion can be completed within less than 1 h. Moreover, the detection limit of the system is found to be 12.00 pg/[micro]L with a high sensitivity up to 90%. Consequently, the proposed saliva-based miniature system can provide a powerful platform for rapid DNA extraction and detection of genetic diseases.

In this work, the ion-transfer voltammetric detection of the protonated [beta]-blocker propranolol in artificial saliva is presented. Cyclic voltammetry, differential pulse voltammetry, and differential pulse stripping voltammetry (DPSV) were employed in the detection of the cationic drug based on ion-transfer voltammetry across arrays of microinterfaces between artificial saliva and an organogel phase. It was found that the artificial saliva matrix decreased the available potential window for ion-transfer voltammetry at this liquid/liquid interface but transfer of protonated propranolol was still achieved. The DPSV method employed a preconditioning step as well as a preconcentration step followed by analytical signal generation based on the back-transfer of the drug across the array of microinterfaces. The DPSV peak current response was linear with drug concentration in the artificial saliva matrix over the concentration range of 0.05-1 [micro]M ([i.sub.p] = -8.13 (nA [micro][M.sup.-1])(concentration) + 0.07 (nA), R = 0.9929, n = 7), and the calculated detection limit (3[S.sub.b]) was 0.02 [micro]M. These results demonstrate that DPSV at arrays of liquid|liquid microinterfaces is a viable analytical approach for pharmaceutical determinations in biomimetic matrixes.

Antibody microarrays have emerged as useful tools for high-throughput protein analysis and candidate biomarker screening. We describe here the development of a multiplexed microsphere-based antibody array capable of simultaneously measuring 10 inflammatory protein mediators. Cytokine-capture microspheres were fabricated by covalently coupling monoclonal antibodies specific for cytokines of interest to fluorescently encoded 3.1 [micro]m polymer microspheres. An optical fiber bundle containing ~50 000 individual 3.1 [micro]m diameter fibers was chemically etched to create microwells in which cytokine-capture microspheres could be deposited. Microspheres were randomly distributed in the wells to produce an antibody array for performing a multiplexed sandwich immunoassay. The array responded specifically to recombinant cytokine solutions in a concentration-dependent fashion. The array was also used to examine endogenous mediator patterns in saliva supernatants from patients with pulmonary inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). This array technology may prove useful as a laboratory-based platform for inflammatory disease research and diagnostics, and its small footprint could also enable integration into a microfluidic cassette for use in point-of-care testing.

Defensins are highly basic cationic peptides that are important components of the innate and adaptive immune response pathways. In addition, these peptides are involved in CD8+ T cell response to HIV-1, increased pulmonary infection risk among cystic fibrosis patients, upregulated levels of HNP-5 for patients with ulcerative colitis and Crohn's disease, and monitoring HNP-3 levels as a tumor classification scheme for cutaneous T cell lymphomas, and have promise in the pharmaceutical field as a new class of antibiotics. Here we present a parallel assay for the ct (HNP1-3) and [beta] (HBD1-2) classes of defensins in saliva that are naturally observed in the concentration range of 1 ng/mL to 10/[micro]g/mL. The method utilizes solid phase extraction of saliva samples combined with liquid chromatography-tandem mass spectrometry to identify and quantitate defensin targets. The approach involves limited sample manipulation and is easily amenable to automation. The saliva samples analyzed are derived from a large cohort study focused on examining the role of polymorphisms in genes of innate and adaptive immunity in modulating the response to vaccination for two gastrointestinal tract infections: typhoid and cholera. The [alpha]-defensin levels observed range from 1 to 10 [micro]g/ mL and correlate well with known active concentrations against a wide variety of pathogens. The observed concentration range for [beta]-defensins was between the detection limit and 33 ng/mL and had a sensitivity level that was comparable to immunoassay-based detection. This method is easily adapted for use in a clinical immunology setting and can be modified for other biological matrixes. This assay will facilitate examination of the production, secretion, and regulation of defensin peptides in a direct fashion to coordinate levels of these compounds with gender, age, response to vaccination, gene copy number, and oral health.

Saliva provides a useful and noninvasive alternative to blood for many biomedical diagnostic assays. The level of the hormone cortisol in blood and saliva is related to the level of stress. We present here the development of a portable surface plasmon resonance (SPR) biosensor system for detection of cortisol in saliva. Cortisol-specific monoclonal antibodies were used to develop a competition assay with a six-channel portable SPR biosensor designed in our laboratory. The detection limit of cortisol in laboratory buffers was 0.36 ng/mL (1.0 nM). An in-line filter based on diffusion through a hollow fiber hydrophilic membrane served to separate small molecules from the complex macromolecular matrix of saliva prior to introduction to the sensor surface. The filtering flow cell provided in-line separation of small molecules from salivary mucins and other large molecules with only a 29% reduction of signal compared with direct flow of the same concentration of analyte over the sensor surface. A standard curve for detection of cortisol in saliva was generated with a detection limit of 1.0 ng/mL (3.6 nM), sufficiently sensitive for clinical use. The system will also be useful for a wide range of applications where small molecular weight analytes are found in complex matrixes.

The foundation for saliva-based diagnostics is the development of a complete catalog of secreted proteins detectable in saliva. Besides protein complexity, the greatest bioanalytical challenge facing comprehensive analysis of saliva samples is related to the large variation of protein relative abundances including the presence of highabundance proteins such as amylases, mucins, prolinerich proteins (PRPs), and secretory IgA complex. Among a number of electrokinetic separation techniques, transient capillary isotachophoresis/capillary zone electrophoresis (CITP/CZE) specifically targets trace amounts of proteins and thus reduces the range of relative protein abundances for providing unparallel advantages toward the identification of low-abundance proteins. By employing a CITP/CZE-based multi-dimensional separation platform coupled with electrospray ionization-tandem mass spectrometry (ESI-tandem MS), a total of 6112 fully tryptic peptides are sequenced at a 1% false discovery rate (FDR), leading to the identification of 1479 distinct human SwissProt protein entries. By comparing with capillary isoelectric focusing (CIEF) as another electrokinetics-based stacking approach, CITP/CZE not only offers a broad field of application but also is less prone to protein/peptide precipitation during the analysis. The ultrahigh resolving power of CITP/CZE is evidenced by the large number of distinct peptide identifications measured from each CITP fraction together with the low peptide fraction overlapping among identified peptides. Furthermore, when evaluating the protein sequence coverage by the number of distinct peptides mapping to each protein identification, the CITP-based proteome technology similarly achieves the superior performance with 674 proteins (46%) having three or more distinct peptides, 288 (19%) having two distinct peptides, and 517 (35%) having a single distinct peptide.