Fold Increase In Sensitivity Research Articles

Cavity-Amplified Scattering Spectroscopy Reveals the Dynamics of Proteins and Nanoparticles in Quasi-transparent and Miniature Samples.

Dynamic light scattering techniques can give access to the motion spectrum of microscopic objects and are therefore routinely used for numerous industrial and research applications ranging from particle sizing to the characterization of the viscoelastic properties of materials. However, such measurements are impossible when samples do not scatter light enough, i.e., when light undergoes too few scattering events when passing through a sample, either due to excessively small scattering cross sections or due to low concentrations of scatterers. Here, we propose to amplify the light scattering efficiency by placing weakly scattering samples inside a Lambertian cavity with high-reflectance walls. When injected with laser light, the cavity produces a 3D isotropic and homogeneous light field, effectively elongates the photon scattering path length through the sample by 2-3 orders of magnitude, and leads to a dramatic increase in sensitivity. With a 104-fold increase in sensitivity compared to classical techniques, we potentially expand the applications of light scattering to miniaturized microfluidics samples and to weakly scattering samples in general. We show that we can access the short-time dynamics of low-turbidity samples and demonstrate our sensitivity gain by measuring the diffusion coefficient and, therefore, the size of particles ranging from 5 nm to 20 μm with volume fractions as low as 10-9 in volumes as low as 100 μL and in solvents with refractive index mismatches down to Δn ≈ 0.01. Beyond the realm of current applications of light scattering techniques, our cavity-amplified scattering spectroscopy method (CASS) and its high sensitivity represent a significant methodological step toward the study of short-time dynamics problems such as the ballistic limit of Brownian motion, the internal dynamics of proteins, or the dielectric dynamics of liquids.

Abstract 1811: Dithiazanine Iodide suppresses mitochondrial function to strongly inhibit pancreatic ductal adenocarcinoma growth in-vitro and in-vivo, producing a marked increase in survival

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive recalcitrant cancer with a uniquely austere tumor microenvironment (TME). It is hypovascularized, hypoxic, and poor in nutrients. These features, which are critical due to the increased energetic demand in continuously replicating cancer cells, drive metabolic reprograming to support the continued activity. Thus leading to a vulnerability in the form of energy restriction - a vulnerability further exacerbated by the nutrient-poor pancreatic cancer TME. We have recently shown mitochondrial inhibition to have great potential in in-vitro inhibition of PDAC cells. Since repurposing of FDA-approved drugs holds great promise in terms of faster and safer drug development, we selected to investigate the effect of mitochondrial inhibition on pancreatic cancer cells through assessment of the previously FDA-approved antimicrobial Dithiazanine Iodide (CDI). CDI is a fluorescent, orally bioavailable, small molecule. In-vitro, it has been previously shown to preferentially localize to the mitochondria, bind proteins and nucleic acids, and have a yet to be discerned inhibitory effect on the electron transport chain. We demonstrated CDI had low IC50s (30-300 nM) in multiple PDAC and other GI cancer cell lines. In an agnostic, unbiased manner, we assessed metabolomic and transcriptomic perturbations caused by CDI. These revealed a profound 92% decrease in the abundance of mitochondrial-encoded transcripts (P<0.05), decreased mitochondrial beta-oxidation, and a reduction in the ATP/ADP ratio. We have therefore hypothesized that the anti-cancer effects of CDI may be related to disruption of mitochondrial processes and we proceeded to characterize its mechanism of action. CDI treatment reduced protein expression of electron transport chain complexes I, II, III, and IV. Treatment with CDI in hypoglycemic conditions resulted in a 3-4 fold increase in sensitivity (P<0.05) and ATP quantification showed a marked >90% decrease in cellular ATP (P<0.05). Furthermore, mitochondria-depleted PDAC cell lines were 4-fold more resistant to CDI treatment (P<0.05). In-vivo, oral CDI treatment resulted in a 2-fold decrease in mouse xenograft tumor growth compared to gemcitabine treatment. RNA-sequencing from the in-vivo samples showed a 60% reduction in the abundance of mitochondrial-encoded transcripts (P<0.05). Examination of the mitochondrial sequences revealed the existence of putative G-Quadruplex sequences. Fluorescence shift and stop-PCR assays confirmed CDI to be a G-Quadruplex binder of mitochondrial sequences. Citation Format: Avinoam Nevler, Christopher W. Schultz, Aditi Jain, Saed Khalilieh, Grace McCarthy, Harish Lavu, Wilbur Bowne, Charles J. Yeo, Jonathan R. Brody. Dithiazanine Iodide suppresses mitochondrial function to strongly inhibit pancreatic ductal adenocarcinoma growth in-vitro and in-vivo, producing a marked increase in survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1811.

Proteomic genotyping: Using mass spectrometry to infer SNP genotypes in a forensic context

Proteins are an intrinsic part of biological evidence. In the last two decades mass spectrometry has revolutionized the analysis of proteins, allowing for thousands of peptides to be detected in a single analysis, including peptides containing single amino acid polymorphisms. Identification of these genetically variant peptides (GVP) allows for inference of the underlying non-synonymous SNP genotype. We have focused on hair proteins as a source of genetic information, discovering and validating 240 GVPs. Using optimized processing, a single hair shaft can obtain random match probabilities (RMPs) of up to 1 in 100 million. We show that GVP-inferred genotypes are not affected by the anatomical origin of the hair shaft or pigmentation. Harsh oxidation with peroxide does not affect RMPs. Importantly, GVP-inferred SNP genotypes are statistically compatible with STR-typing and Alu retroelements, with 90% of GVP-inferred SNPs being located greater than 10 million and 75 million bp from the nearest STR or Alu element respectively. We have shown separation of DNA and peptide workflows from the same samples. Recent work has shifted to the use of a targeted peptide assay. When data acquisition of GVPs was dynamically triggered by the detection of a standard peptide, this approach resulted in a 2.5 fold increase in sensitivity and increased confidence in peptide detection. Based on Monte Carlo modeling, we predict this increase in sensitivity will result in RMPs in excess of 1 in 1 trillion from a single hair shaft. Proteomic genotyping can also be applied to any protein matrix, including fingermarks and bone. Proteomic genotyping therefore has potential to complement partial PCR-based DNA typing and to provide other options for investigators.

Method of Immuno-PCR in diagnostics of bacterial and viral infections

Immuno-PCR (I-PCR) combines the capabilities of two modern diagnostic methods of enzyme immunoassay (ELISA) and polymerase chain reaction (PCR), combination these methods causes a 100-10000 fold increase in sensitivity compared to a similar ELISA. The review considers the main variants of I-PCR, gives examples of possible use of the method for early and retrospective diagnosis of various diseases.

Targeting a highly repeated germline DNA sequence for improved real-time PCR-based detection of Ascaris infection in human stool.

BackgroundWith the expansion of soil transmitted helminth (STH) intervention efforts and the corresponding decline in infection prevalence, there is an increased need for sensitive and specific STH diagnostic assays. Previously, through next generation sequencing (NGS)-based identification and targeting of non-coding, high copy-number repetitive DNA sequences, we described the development of a panel of improved quantitative real-time PCR (qPCR)-based assays for the detection of Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Trichuris trichiura, and Strongyloides stercoralis. However, due to the phenomenon of chromosome diminution, a similar assay based on high copy-number repetitive DNA was not developed for the detection of Ascaris lumbricoides. Recently, the publication of a reference-level germline genome sequence for A. lumbricoides has facilitated our development of an improved assay for this human pathogen of vast global importance.Methodology/Principal findingsRepurposing raw DNA sequence reads from a previously published Illumina-generated, NGS-based A. lumbricoides germline genome sequencing project, we performed a cluster-based repeat analysis utilizing RepeatExplorer2 software. This analysis identified the most prevalent repetitive DNA element of the A. lumbricoides germline genome (AGR, Ascaris germline repeat), which was then used to develop an improved qPCR assay. During experimental validation, this assay demonstrated a fold increase in sensitivity of ~3,100, as determined by relative Cq values, when compared with an assay utilizing a previously published, frequently employed, ribosomal internal transcribed spacer (ITS) DNA target. A comparative analysis of 2,784 field-collected samples was then performed, successfully verifying this improved sensitivity.Conclusions/SignificanceThrough analysis of the germline genome sequence of A. lumbricoides, a vastly improved qPCR assay has been developed. This assay, utilizing a high copy-number repeat target found in eggs and embryos (the AGR repeat), will improve prevalence estimates that are fundamental to the programmatic decision-making process, while simultaneously strengthening mathematical models used to examine STH infection rates. Furthermore, through the identification of an optimal target for PCR, future assay development efforts will also benefit, as the identity of the optimized repeat DNA target is likely to remain unchanged despite continued improvement in PCR-based diagnostic technologies.

DDIS-05. TARGETING MESENCHYMAL GLIOBLASTOMA THROUGH INHIBITION OF DIACYLGLYCEROL KINASE ALPHA

Recent studies have shown that glioblastoma (GBM) can be classified into distinct subtypes based on gene expression signatures: classical, proneural, and mesenchymal. Of these, the mesenchymal subtype is relatively more aggressive and resistant to therapy, and is thought to be a primary driver of recurrence. The diacylglycerol kinase alpha (DGKα) signaling pathway has extensive connections to oncogenic pathways/proteins. Recently, we identified ritanserin, an abandoned serotonin receptor 5-HT2 inhibitor originally developed for schizophrenia, as a novel DGKα inhibitor. Compared to a known DGKα inhibitor, R59022, ritanserin is more potent and has favorable pharmacologic features such as long plasma half-life and known BBB penetration. Based on the TCGA database, we have determined that DGKα expression level is highest in the mesenchymal subtype. DGKα inhibition with ritanserin revealed a significant difference in IC50 values, with a 3-4 fold increase in sensitivity, of all mesenchymal GBM stem cell (GSC) lines tested versus all non-mesenchymal lines. In vivo, daily oral ritanserin increased mice survival significantly with two mesenchymal GSC lines, while there was no survival benefit with a proneural GSC line. With an EMT model, both lung and pancreatic carcinoma cells became more sensitive to ritanserin after the acquisition of mesenchymal phenotype. Ritanserin reduces NF-κB activity significantly and suppresses geranylgeranylation of proteins by inhibiting geranylgeranyltransferase I. Exogenous phosphatidic acid, the DGKα enzymatic product, substantially rescued GBM cells from ritanserin toxicity while exogenous 5-HT2 receptor agonist, 5-hydroxytryptamine, showed a small rescue from ritanserin toxicity. In keeping with its anti-mesenchymal activity, ritanserin functions as a radiosensitizer, with suppression of the DNA damage response and shows significant synergy with radiation. Our work is the first to show in vitro and in vivo that a clinically practical agent has strong preferential toxicity for the mesenchymal GBM subtype, with potential therapeutic implications for GBM and other cancers as well.

Mesoporous nickel cobalt hydroxide/oxide as an excellent room temperature ammonia sensor

We report an excellent room temperature NH3 gas sensing performance using highly mesoporous nanoflakes of Ni-Co hydroxide/Oxide synthesised by facile and economical wet chemical method. The dynamic sensing of NiCo2(OH)6(NCH) and NiCo2O4 (NCO) shows nearly 3 and 2 fold increase in sensitivity by varying NH3 concentrations demonstrating excellent sensitivity which is in accordance to Freundlich equation. The interesting behaviour of NCO being p–type material spectacles n-type gas sensing behaviour towards low ammonia concentration at RT. The anomalous sensing response is ascribed to transition of majority charge carriers due to surface reaction of chemisorbed oxygen ions and ammonia gas vapours.

Abstract B27: First-in-class cytoskeletal targeting drug, Anisina, enhances effectiveness of microtubule inhibitors in a preclinical model of neuroblastoma

Abstract Neuroblastoma is the most common extracranial solid tumor in childhood accounting for ~7% of all cancer cases in children. Children with high-risk neuroblastoma still have a poor overall outcome when treated with existing chemotherapeutics, making it imperative that new chemotherapeutic strategies are developed to treat this disease. The actin cytoskeleton is an ideal chemotherapeutic target due to its role in numerous biological processes essential for tumor cell growth and survival. While the therapeutic potential of actin-targeting compounds has been extensively investigated, no lead compound has been developed through to the clinic due to the associated cardiac and respiratory toxicity. We have developed a novel class of anti-tropomyosin (ATM) drugs that target cancer- associated actin filaments containing the tropomyosin Tpm3.1. The lead ATM, Anisina, was identified utilizing Novogen's proprietary VAL-ID - Versatile Approach to Library-based Iterative Design – medicinal chemistry program. This strategy is based around the design, synthesis and evaluation of targeted small-molecule libraries and has proven to be a rapid and robust method of identifying lead compounds. In vitro studies have demonstrated that Anisina binds to and impairs the function of Tpm3.1 in a dose dependent manner in both protein and cell based assays. Inhibition of Tpm3.1 function in a tumor cell model results in the destabilization of Tpm3.1 containing actin filaments and induction of tumor cell death via the activation of the intrinsic apoptotic pathway. Anisina has broad efficacy against both adult and pediatric cancers in vitro. When tested against a panel of neuroblastoma cell lines, Anisina was effective independent of NMYC status and had an average IC50 of 4-7uM. In preparation for the clinic, in vitro synergy studies were undertaken to evaluate the impact of Anisina in combination with standard of care chemotherapeutics. Anisina showed a high degree of synergy (combinatorial index <0.5) with anti-microtubule agents, both vinca alkaloids (microtubule destabilizing) and taxanes (microtubule stabilizing) in some adult and pediatric cancer cell models. In particular a ~20 fold increase in sensitivity to drug induced cell death was observed in tumor cells treated with the Anisina/Vincristine (VCR) combination. The same combinatorial effect was not observed with untransformed cells. More importantly, the synergy observed with Anisina/VCR in vitro has now been replicated in a CHLA20 human neuroblastoma xenograft model. Animals (n=4-5) were randomly divided into four treatment groups i) Control (Captisol), ii) Anisina (150mg/kg daily IP) iii) VCR alone (0.5mg/kg weekly, IV) and iv) Anisina (150mg/kg) + VCR (0.5mg/kg). Animals treated with Anisina/VCR showed a significant and profound regression of tumor growth compared to control and either drug treatment alone. Further to this, of the 5 animals in the combinatorial Anisina/VCR treatment group 3 (60%) reached a “complete response” with one animal showing a “maintained complete response” for more than 100 days post treatment. The outcomes of this study have significant therapeutic implications as microtubule inhibitors such as VCR are current standard of care for a wide range of solid tumors, including neuroblastoma. Anisina is currently being developed as an adjunct therapy for the treatment of both adult and pediatric cancers. Citation Format: Justine Stehn, Mark Currier, Duo Chen, Eleanor Eiffe, Andrew Heaton, Jeff Hook, Peter Gunning, Timothy Cripe. First-in-class cytoskeletal targeting drug, Anisina, enhances effectiveness of microtubule inhibitors in a preclinical model of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B27.

Editorial

We present a novel technique aimed at improving upon the thermoluminescence (TL) response of optical fibers. The technique, based on the stack-and-draw method, is more conventionally used for microstructured optical fiber (MOF) fabrication. Utilizing the approach, the TL response of a single microstructured fiber can be shown to substantially improve upon that of a single capillary fiber, approaching a 30 fold increase in sensitivity. Present results provide strong support for the idea that by collapsing and fusing the surface walls of stacked fibers, strain-related defects are created, increasing the TL yield many times over. The substantial increase in sensitivity of these glass-based systems point to more extensive applications, the production of such detectors allowing versatile use, down to much lower doses than currently available using single capillary fibers.

Is an increased drop-in rate appropriate with enhanced DNA profiling?

With enhanced capillary electrophoresis (CE) injection the fold increase in peak heights relates to the fold increase in sensitivity. As a result, DNA profiles appear similar when the amount of DNA in the PCR was adjusted according to the sensitivity of the analysis. With enhanced CE injection, however, more drop-in alleles were observed. In (probabilistic) DNA profile interpretation, it seems opportune to account for an amount of drop-in that is in accordance with the sensitivity of the method.

Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum

Candida albicans is an opportunistic pathogen which can lead to Candidiasis and blood-stream infections, resulting in a mortality rate near 40%. Given its high fatality and emerging pathogenicity, there is a strong need for the development of a rapid C. albicans diagnostic assay. Point-of-care devices, specifically lateral flow assays, are an attractive and often employed diagnostic modality for C. albicans detection. However, they lack the required performance characteristics needed for accurate pathogen detection and subsequent treatment options. Thus, we describe herein the utility of the Dual Path Platform (DPP®) device as an immunochromatographic Point-of-care assay for C. albicans. The limit of detection for hyphal and budding C. albicans in DPP® tests are reported to be as low as 7.94×105 whole cells/mL in human serum. C. albicans cells were detected with up to a 3.9 fold increase in sensitivity on DPP® when compared to conventional lateral flow modalities.

Improving thermoluminescence response through the fabrication of novel microstructured fibers

Abstract We present a novel technique aimed at improving upon the thermoluminescence (TL) response of optical fibers. The technique, based on the stack-and-draw method, is more conventionally used for microstructured optical fiber (MOF) fabrication. Utilizing the approach, the TL response of a single microstructured fiber can be shown to substantially improve upon that of a single capillary fiber, approaching a 30 fold increase in sensitivity. Present results provide strong support for the idea that by collapsing and fusing the surface walls of stacked fibers, strain-related defects are created, increasing the TL yield many times over. The substantial increase in sensitivity of these glass-based systems point to more extensive applications, the production of such detectors allowing versatile use, down to much lower doses than currently available using single capillary fibers.

The Risk of Relapse in CML Patients Who Discontinued imatinib Can Be Predicted Based on Patients Age and the Results of dPCR Analysis

▪Introduction. Chronic myeloid leukemia (CML) patients (pts) treated with imatinib first line achieve complete cytogenetic response (CCyR) in > 70% of cases and major molecular response (MMR) in 18-58%. These pts have a life expectancy similar to the general population. However even undetectable BCR-ABL may not equate to eradication of the disease because of the sensitivity of Q-RT-PCR. A new diagnostic method, the digital-PCR (dPCR), able to detect 1 BCR-ABL+ cell out of 107 cells, has been recently developed (Goh HG et al., 2011). dPCR corresponds to a 100 fold increase in sensitivity as compared to Q-RT-PCR. Therefore, dPCR by assessing the presence of minimal residual disease with higher sensitivity, could potentially identify pts in whom CML has been eradicated.Aims. The Imatinib Suspension And Validation (ISAV) study is aimed at validating the capability of dPCR to predict relapses after imatinib discontinuation in CML pts with negative Q-RT-PCR results.Methods. This study involves 15 sites, 10 in Italy and 1 in each of the following countries: Germany, Spain, The Netherlands, Canada and Israel. CML pts (Chronic or Accelerated Phase) under imatinib therapy since more than 2 years and in complete molecular remission (CMR) were eligible for this study. Patients had to be in CMR for at least 18 months (mts), with a minimum of 3 Q-RT-PCR performed at their own sites. After signing the informed consent, blood samples are obtained for dPCR and the pts discontinue imatinib therapy. Standard Q-RT-PCR is performed monthly (mts 1-6) and then bimonthly for 36 mts to assess the maintenance of the molecular remission. The loss of molecular remission is defined as two consecutive positive Q-RT-PCR tests with at least one BCR-ABL/ABL value above 0.1%. Patients losing molecular remission resume imatinib treatment at the same dosage used before interruption. Patients’ quality of life during imatinib discontinuation/resumption is evaluated through the EORTC – C30 Quality of Life questionnaire.Results. The enrolment in ISAV began in November 2011 and ended in July 2013. The study enrolled 112 pts: Italy 69.6%, Germany 21.4%, Canada 5.3%, Spain 2.6% and Israel 0.9%. Among the 112 pts, 59.3% were male and 37.0% were aged 65 or older; median duration of imatinib treatment was 103.1 mts with median duration of CMR of 25.8 mts before imatinib discontinuation. To date, the median follow-up (FUP) time is 16.6 mts [95% CI: 14.9-18.2]. Forty-seven pts (43.5%, 95% CI: 34.0-53.4) of the 108 eligible pts relapsed and resumed imatinib; 38/47 (80.9%) of them relapsed in the first 9 mts and the last relapse occurred 19.6 mts after imatinib discontinuation. A loss of CCyR occurred in 11 pts (23.4%): 10/11 CCyR losses were recovered; 1 patient withdrew the consent shortly after obtaining a partial cytogenetic response. No case of CML progression was observed. After the resumption of imatinib the median time to either MMR or CMR was 1.9 [95% CI: 1.2-2.4] mts. Of the 61 not-relapsed pts, 43 (39.8% of the total) regained Q-RT-PCR positivity but never lost MMR. The median time to Q-RT-PCR positivity was 3.6 mts [95% CI: 3.0-4.8] and the range of duration of Q-RT-PCR positivity (below 0.1%) was between 5.7 and 29.2 mts. No significant correlation between relapse and previous duration of imatinib treatment, use of interferon, time to CCyR or duration of CMR was identified. An inverse relationship between pts age and risk of relapse is evident: 90% of pts < 45 years relapsed vs 37.5% in the class ≥ 45 - < 65 years and 27.5% of pts ≥ 65 years, p(χ2)<0.0001. dPCR results showed that 23.4% of pts were positive and 76.6% negative, with a dPCR Negative Predictive Value (NPV) of 63.4% (Tab.1) and a significant NPV ratio (dPCR/Q-RT-PCR) of 1.131 [95% CI: 1.032-1.239]. Age and dPCR results predicted the risk of relapse: pts with less than 45 years and with a positive dPCR had the highest risk of relapse (100%) as opposed to pts ≥ 45 years and with negative dPCR (30.6%; Fig.1).Conclusions. After 32 mts from the beginning of the study, with a median FUP of 16.6 mts, 43.5% of pts relapsed; the majority of relapses developed in the first 9 months after imatinib discontinuation. Age < 45 years and dPCR positivity are significantly associated with relapses. Funded by Regione Lombardia. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The use of a polymer inclusion membrane as a sorbent for online preconcentration in the flow injection determination of thiocyanate impurity in ammonium sulfate fertilizer

A polymer inclusion membrane (PIM) is used for the first time as a sorbent in the construction of a preconcentration column to enhance the sensitivity in flow injection analysis (FIA). The PIM-coated column is readily prepared by coating the PIM containing poly(vinyl chloride), Aliquat 336, and 1-tetradecanol onto glass beads packed in a glass tube. The determination of trace amounts of thiocyanate in ammonium sulfate fertilizer demonstrates the potential of the proposed PIM-coated column in FIA. Thiocyanate standards or samples of relatively large volume (e.g. up to 2000µL) are injected into a nitrate carrier stream. The sample zone passes through the proposed preconcentration column where thiocyanate is concentrated in a smaller volume of a carrier solution thus resulting in up to 7.4 fold increase in sensitivity. Thiocyanate is detected spectrophotometrically after its reaction with Fe(III) downstream of the preconcentration column. The limits of detection of thiocyanate in the absence and presence of 20gL−1 ammonium sulfate (S/N=2) are 0.014 and 0.024mgL−1, respectively. Thiocyanate was successfully determined in several samples of ammonium sulfate fertilizer.

Fabrication of 1D ZnO nanostructures on MEMS cantilever for VOC sensor application

This study reports the fabrication method and sensing performance for novel 1D zinc oxide (ZnO) nanorods and nanotubes grown on nickel MEMS cantilevers. The fabrication of the nanostructures and the cantilevers are simple and low-cost using standard lithography, electrodeposition, and hydrothermal etching processes. 1D ZnO nanostructures increase the total sensitive area for biological and chemical sensor applications. We performed experiments with various VOCs with a real-time sensor system developed in our laboratory. While Ni microcantilevers produced no signal, ZnO nanostructure coated microcantilevers showed good sensitivity and repeatable changes. Furthermore, the nanotube coated microcantilevers showed more than 10 fold increase in sensitivity compared to the nanorod coated microcantilevers which can be explained to the fact that ZnO nanotubes have higher surface area and subsurface oxygen vacancies and these provide a larger effective surface area with higher surface-to-volume ratio as compared to ZnO nanorods. The tests are performed using dynamic mode of operation near resonant frequency using magnetic actuation and optical sensing. The phase stability and the limit of detection of ZnO nanotube coated microcantilevers exposed to diethylamine (DEA) were 0.02° and lower than 10ppm, respectively. ZnO nanostructure coated microcantilevers have good potential for VOC sensor applications especially for amine groups.

Theoretical modeling of lossy mode resonance based refractive index sensors with ITO/TiO₂ bilayers.

A lossy mode resonance (LMR) based fiber optic refractive index (RI) sensor utilizing indium tin oxide (ITO)/titanium dioxide (TiO₂) bilayers has been theoretically investigated. Specifically, the effect of different thickness ratios of bilayers on the sensitivity to surrounding medium RI variations was studied. It was observed that for a specific thickness ratio, a two fold increase in sensitivity can be achieved in comparison to conventional LMR sensors using a single absorbing thin film. The sensitivity can be enhanced by tuning the total bilayer thickness and bilayer thickness ratio for the desired application, which is not possible in conventional LMR sensors.

A highly stable, sensitive, regenerable and rapid immunoassay for detecting aflatoxin B1 in corn incorporating covalent AFB1 immobilization and a recombinant Fab antibody

A highly robust immunoassay applicable for the detection of aflatoxin B1 (AFB1) using a Fab antibody fragment was developed. A key factor was the use of covalently immobilized AFB1 which allowed an almost three fold increase in sensitivity, reduced assay time and regeneration with retention of binding capacity. Various factors that might affect the sensitivity of the assay such as pH, organic solvents, storage stability and wash stringency were critically evaluated. It was also demonstrated that the assay was applicable for determination of AFB1 in corn samples at concentration within the European union regulatory limits.

Helicobacter pylori versus the Host: Remodeling of the Bacterial Outer Membrane Is Required for Survival in the Gastric Mucosa

Modification of bacterial surface structures, such as the lipid A portion of lipopolysaccharide (LPS), is used by many pathogenic bacteria to help evade the host innate immune response. Helicobacter pylori, a gram-negative bacterium capable of chronic colonization of the human stomach, modifies its lipid A by removal of phosphate groups from the 1- and 4′-positions of the lipid A backbone. In this study, we identify the enzyme responsible for dephosphorylation of the lipid A 4′-phosphate group in H. pylori, Jhp1487 (LpxF). To ascertain the role these modifications play in the pathogenesis of H. pylori, we created mutants in lpxE (1-phosphatase), lpxF (4′-phosphatase) and a double lpxE/F mutant. Analysis of lipid A isolated from lpxE and lpxF mutants revealed lipid A species with a 1 or 4′-phosphate group, respectively while the double lpxE/F mutant revealed a bis-phosphorylated lipid A. Mutants lacking lpxE, lpxF, or lpxE/F show a 16, 360 and 1020 fold increase in sensitivity to the cationic antimicrobial peptide polymyxin B, respectively. Moreover, a similar loss of resistance is seen against a variety of CAMPs found in the human body including LL37, β-defensin 2, and P-113. Using a fluorescent derivative of polymyxin we demonstrate that, unlike wild type bacteria, polymyxin readily associates with the lpxE/F mutant. Presumably, the increase in the negative charge of H. pylori LPS allows for binding of the peptide to the bacterial surface. Interestingly, the action of LpxE and LpxF was shown to decrease recognition of Helicobacter LPS by the innate immune receptor, Toll-like Receptor 4. Furthermore, lpxE/F mutants were unable to colonize the gastric mucosa of C57BL/6J and C57BL/6J tlr4 -/- mice when compared to wild type H. pylori. Our results demonstrate that dephosphorylation of the lipid A domain of H. pylori LPS by LpxE and LpxF is key to its ability to colonize a mammalian host.

Effect of processable polyindole and nanostructured domain on the selective sensing of dopamine

The effect of carboxylic acid functionality present in polymer backbone is reported on electrochemical sensing of dopamine (DA). The electropolymerized conducting polymers made from carboxylic acid substituted indole at positions − 5 and − 6 are found processable in aqueous medium and are compatible with suitable additives/precursors for fabricating polymer modified electrodes (PMEs). The modified electrodes are fabricated following two methods, i.e.: (1) the processable polymers are cast over glassy carbon electrode (GCE) using Nafion® followed by chemical modification using hydrophobic organic redox mediators and (2) the processable polymers are encapsulated within organically modified silicate (Ormosil) matrix along with the hydrophilic redox mediator followed by incorporation of silver and gold nanoparticles. The electrochemical performances of these modified electrodes show selective sensing of DA with major findings: (i) both polymers introduced selectivity in electrochemical sensing of DA with analogous sensitivity, (ii) sensitivity is enhanced when hydrophobic organic redox mediators are coupled with modified electrode matrix involving Nafion®, (iii) the polymers are suitable for encapsulation within ormosil matrix thus introducing nanostructured network for further improvement in sensitivity of DA analysis, (iv) the presence of gold and silver nanoparticles within ormosil matrix along with polymers caused > 100 fold increase in sensitivity of DA sensing with lowest detection limit to the order of 100 nM.

Optimization of a real-time RT-PCR assay reveals an increase of genogroup I norovirus in the clinical setting

Although norovirus has been identified as the most common cause of gastroenteritis, the majority of cases have no etiologic agent identified. In this study, we describe the optimization of a real-time RT-PCR assay for the improved detection of genogroup I norovirus in patient specimens based upon sequence data from a collection of representative clinical norovirus sequences. The redesigned assay demonstrated a 64 fold increase in sensitivity, a 2 log decrease in the limit of detection, and an 18% increase in amplification efficiency, when compared to the standard assay. The optimized test also detected GI norovirus in clinical specimens that were initially negative by the standard assay. Use of the optimized assay increased the annual positivity of GI norovirus in Iowa from 1.2% to 4.5%, indicating the prevalence of GI norovirus may be higher than previously identified. Laboratory confirmation of the etiologic agent involved in gasteroenteritis cases is essential for better understanding of the prevalence and transmission of noroviruses.