Semiautomated Electrochemical Melting Curve Analysis Device for the Detection of an Osteoporosis Associated Single Nucleotide Polymorphism in Blood.

The detection of single nucleotide polymorphisms (SNPs) is of increasing importance in many areas including clinical diagnostics, patient stratification for pharmacogenomics, and advanced forensic analysis. In the work reported, we apply a semiautomated system for solid-phase electrochemical melting curve analysis (éMCA) for the identification of the allele present at a specific SNP site associated with an increased risk of bone fracture and predisposition to osteoporosis. Asymmetric isothermal recombinase polymerase amplification using ferrocene labeled forward primers was employed to generate single stranded redox labeled amplicons. In a first approach to demonstrate the proof of concept of combining asymmetric RPA with solid-phase éMCA, a simplified system housing a multielectrode array within a polymeric microsystem, sandwiched between two aluminum plates of a heater device, was used. Sample manipulation through the microfluidic channel was controlled by a syringe pump, and an external Ag/AgCl reference electrode was employed. Individual electrodes of the array were functionalized with four different oligonucleotide probes, each probe equivalent in design with the exception of the middle nucleotide. The isothermally generated amplicons were allowed to hybridize to the surface-tethered probes and subsequently subjected to a controlled temperature ramp, and the melting of the duplex was monitored electrochemically. A clear difference between the fully complementary and a single mismatch was observed. Having demonstrated the proof-of-concept, a device for automated éMCA with increased flexibility to house diverse electrode arrays with internal quasi-gold reference electrodes, higher resolution, and broader melting temperature range was developed and exploited for the detection of SNP hetero/homozygosity. Using the optimized conditions, the system was applied to the identification of the allele present at an osteoporosis associated SNP site, rs2741856, in 10 real fingerprick/venous blood samples, with results validated using Sanger sequencing.

Novel nandrolone aptamer for rapid colorimetric detection of anabolic steroids.

The illicit use of anabolic androgenic steroids (AAS) as performance-enhancing drugs remains a global issue threatening not only the credibility of competitive sports but also public health due to the well-documented adverse effects they elicit. AAS abuse is not restricted only to professional sports, but also extends to recreational athletes and adolescents as well as in livestock production as growth-promoting agents. Testosterone and nandrolone are among the AAS most frequently exploited. Gas chromatography-mass spectrometry is the reference method for AAS detection, but it is strictly laboratory-based and cannot be performed on-site. The great potential of aptamers in bioanalytical applications and specifically for the development of simple analytical tools suitable for on-site analysis has been extensively documented. In this report, we describe the selection and identification of aptamers binding nandrolone, exhibiting affinity dissociation constants in the low nanomolar range. A label-free colorimetric assay based on gold nanoparticles was developed using one of these novel aptamers for the detection of nandrolone and/or its metabolites. The assay could be deployed for the rapid, on-site, facile and cost-effective screening of samples and provide qualitative visual results with a red to purple/blue color change being indicative of a positive result.

Solid-Phase Primer Elongation Using Biotinylated dNTPs for the Detection of a Single Nucleotide Polymorphism from a Fingerprick Blood Sample.

Isothermal recombinase polymerase amplification-based solid-phase primer extension is used for the optical detection of a hypertrophic cardiomyopathy associated single nucleotide polymorphism (SNP) in a fingerprick blood sample. The assay exploits four thiolated primers which have the same sequences with the exception of the 3'-terminal base. Target DNA containing the SNP site hybridizes to all four of the immobilized probes, with primer extension only taking place from the primer containing the terminal base that is complementary to the SNP under interrogation. Biotinylated deoxynucleotide triphosphates are used in the primer extension, allowing postextension addition of streptavidin-poly-horseradish peroxidase to bind to the incorporated biotinylated dNTPs. The signal generated following substrate addition can then be measured optically. The percentage of biotinylated dNTPs and the duration of primer extension is optimized and the system applied to the identification of a SNP in a fingerprick blood sample. A methodology of thermal lysis using a 1 in 5 dilution of the fingerprick blood sample prior to application of 95 °C for 30 s is used to extract genomic DNA, which is directly used as a template for solid-phase primer extension on microtiter plates, followed by optical detection. The SNP in the fingerprick sample was identified and its identity corroborated using ion torrent next generation sequencing. Ongoing work is focused on extension to the multiplexed detection of SNPs in fingerprick and other biological samples.

A longitudinal study monitoring the quality of life in a national cohort of older adults in Chile before and during the COVID-19 outbreak.

BACKGROUND: Confinement during the COVID-19 pandemic has placed great stress on older adults, which may be affecting their quality of life. Thus, this study aims to describe the changes in mental and physical health, isolation and loneliness, residence and socioeconomic resources in a national cohort of Chilean older adults before and during the COVID-19 outbreak. It also analyzes the changes in depressive symptoms by changes in the other quality of life indicators before and during the COVID-19 outbreak. Possible methodological biases of telephone surveys in older adults living in non-developed countries are also discussed. METHODS: Between June and September 2020, a random subsample of 720 people who had participated in the face-to-face V National Survey on Quality of Life in Older Adults in Chile conducted at the end of 2019 was followed up by telephone. Descriptive bivariate analyses were performed using t-test and non-parametric tests for independent variables, comparing the baseline sample with the current 2020 follow-up sample during the peak of the pandemic outbreak in Latin America. Furthermore, descriptive bivariate analysis through t-test and non-parametric test for paired samples compared the follow-up subsample at baseline with the not-included sample, examining possible biases of the telephone interview compared with the face-to-face interview. RESULTS: In the panel, there was no variation in self-rated health. The health symptoms that worsened were memory, stomach, and mood problems. Depressive symptoms and anxiety increased; similarly, smartphone users, social contacts, intergenerational co-residence and resilience increased. The telephone follow-up sample had a higher educational level and greater smartphone use than those not included in the subsample. CONCLUSIONS: Although some physical and mental health indicators have worsened during the pandemic, older adults mobilized resources that could allow them to maintain their quality of life, such as improved resilience. Thus, these findings can guide future research and the development of efficient strategies to improve these resources among older adults to ensure wellbeing.

Aptamers against the ß-Conglutin Allergen: Insights into the Behavior of the Shortest Multimeric (Intra)Molecular DNA G-Quadruplex.

In previous work, a 93-mer aptamer was selected against the anaphylactic allergen, ß-conglutin and truncated to an 11-mer, improving the affinity by two orders of magnitude, whilst maintaining the specificity. This 11-mer was observed to fold in a G-quadruplex, and preliminary results indicated the existence of a combination of monomeric and higher-order structures. Building on this previous work, in the current study, we aimed to elucidate a deeper understanding of the structural forms of this 11-mer and the effect of the structure on its binding ability. A battery of techniques including polyacrylamide gel electrophoresis, high-performance liquid chromatography in combination with electrospray ionization time-of-flight mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight, thermal binding analysis, circular dichroism and nuclear magnetic resonance were used to probe the structure of both the 11-mer and the 11-mer flanked with TT- at either the 5' or 3' end or at both ends. The TT-tail at the 5' end hinders stacking effects and effectively enforces the 11-mer to maintain a monomeric form. The 11-mer and the TT- derivatives of the 11-mer were also evaluated for their ability to bind its cognate target using microscale thermophoresis and surface plasmon resonance, and biolayer interferometry confirmed the nanomolar affinity of the 11-mer. All the techniques utilized confirmed that the 11-mer was found to exist in a combination of monomeric and higher-order structures, and that independent of the structural form present, nanomolar affinity was observed.

High Affinity Aptamer for the Detection of the Biogenic Amine Histamine.

The importance of histamine in various physiological functions and its involvement in allergenic responses make this small molecule one of the most studied biogenic amines. Even though a variety of chromatography-based methods have been described for its analytical determination, the disadvantages they present in terms of cost, analysis time, and low portability limit their suitability for in situ routine testing. In this work, we sought to identify histamine-binding aptamers that could then be exploited for the development of rapid, facile, and sensitive assays for histamine detection suitable for point-of-need analysis. A classic SELEX process was designed employing magnetic beads for target immobilization and the selection was completed after ten rounds. Following Next Generation Sequencing of the last selection rounds from both positive and counter selection magnetic beads, several sequences were identified and initially screened using an apta-PCR affinity assay (APAA). Structural and functional characterization of the candidates resulted in the identification of the H2 aptamer. The high binding affinity of the H2 aptamer to histamine was validated using four independent assays ( KD of 3-34 nM). Finally, the H2 aptamer was used for the development of a magnetic beads-based competitive assay for the detection of histamine in both buffer and synthetic urine, achieving very low limits of detection of 18 pM and 76 pM, respectively, while no matrix effects were observed. These results highlight the suitability of the strategy followed for identifying small molecule-binding aptamers and the compatibility of the selected H2 aptamer with the analysis of biological samples, thus facilitating the development of point-of-care devices for routine testing. Ongoing work is focused on extending the application of the H2 aptamer to the detection of spoilage in meat, fish, and beverages, as well as evaluating the affinity of truncated forms of the aptamer.

Impact of mind-body interventions in older adults with mild cognitive impairment: a systematic review.

ABSTRACTBackground:Mind-body interventions have been associated with a range of positive outcomes in older adults with mild cognitive impairment (MCI). The aim of the present study was to review the impact of different non-pharmacological programs based on mind-body intervention for older adults with MCI. METHODS: A comprehensive search method as required by the Cochrane Collaboration has been performed through the following databases: Google Scholar, Science Direct, PubMed, PsycINFO, MEDLINE, EMBASE, CINHAL, Cochrane, Ebsco. We included the studies that evaluated the impact of mind-body interventions such as mindfulness or meditation, yoga, Tai Chi and Qigong on cognitive function and everyday functionality of non-hospitalized adults aged 55 years or over with MCI. RESULTS: Nine studies met the inclusion criteria. Results indicated that mind-body interventions improved cognitive function, everyday activities functioning, and mindfulness, as well as resulting in a moderate reduction in fall risk, depression and stress and lower risk of dementia at one year. CONCLUSION: Several mind-body interventions focused broadly on mindfulness, yoga and Tai Chi training have been studied. This review shows that mind-body interventions improved cognitive function and everyday activities functioning, memory, resilience and mindfulness in older adults with MCI. However, the conclusions faced limitations, such as small sample size, heterogeneity of outcome measures, lack of an active control group and absence of long-term follow up. Further high-quality evidence is needed in order to determine whether mind-body interventions are cost-effective for improving cognitive decline in older adults with MCI and for delaying the rapid progression from MCI to Alzheimer or other types of dementia.

Duplex Lateral Flow Assay for the Simultaneous Detection of Yersinia pestis and Francisella tularensis.

High-risk pathogens such as Francisella tularensis and Yersinia pestis are categorized as highly hazardous organisms that can be used as biological weapons. Given the extreme infectivity of these potential biowarfare agents, a rapid, sensitive, cost-effective, and specific method for their detection is required. Here, we report the multiplexed amplification detection of genomic DNA from Francisella tularensis and Yersinia pestis. Amplification was achieved using isothermal recombinase polymerase amplification, exploiting tailed primers, followed by detection using a nucleic-acid lateral flow assay. Excess primers were removed using a novel fishing strategy, avoiding the use of postamplification purification that requires centrifugation and infers additional assay cost. The entire assay is completed in less than 1 h, achieving limits of detection of 243 fg (1.21 × 102 genome equivalent) and 4 fg (0.85 genome equivalent) for Francisella tularensis and Yersinia pestis, respectively.

Isothermal amplification using modified primers for rapid electrochemical analysis of coeliac disease associated DQB1*02 HLA allele.

DNA biosensors are attractive tools for genetic analysis as there is an increasing need for rapid and low-cost DNA analysis, primarily driven by applications in personalized pharmacogenomics, clinical diagnostics, rapid pathogen detection, food traceability and forensics. A rapid electrochemical genosensor detection methodology exploiting a combination of modified primers for solution-phase isothermal amplification, followed by rapid detection via hybridization on gold electrodes is reported. Modified reverse primers, exploiting a C18 spacer between the primer-binding site and an engineered single stranded tail, are used in a recombinase polymerase amplification reaction to produce an amplicon with a central duplex flanked by two single stranded tails. These tails are designed to be complementary to a gold electrode tethered capture oligo probe as well as a horseradish peroxidase labelled reporter oligo probe. The time required for hybridization of the isothermally generated amplicons with each of the immobilized and reporter probes was optimised to be 2 min, in both cases. The effect of amplification time and the limit of detection were evaluated using these hybridization times for both single stranded and double stranded DNA templates. The best detection limit of 70 fM for a ssDNA template was achieved using 45 min amplification, whilst for a dsDNA template, just 30 min amplification resulted in a slightly lower detection limit of 14 fM, whilst both 20 and 45 min amplification times were observed to provide detection limits of 71 and 72 fM, respectively, but 30 and 45 min amplification resulted in a much higher signal and sensitivity. The genosensor was applied to genomic DNA and real patient and control blood samples for detection of the coeliac disease associated DQB1*02 HLA allele, as a model system, demonstrating the possibility to carry out molecular diagnostics, combining amplification and detection in a rapid and facile manner.

Antimicrobial lock solutions for preventing catheter-related infections in haemodialysis.

BACKGROUND: Patients undergoing haemodialysis (HD) through a central venous catheter (CVC) are exposed to several risks, being a catheter-related infection (CRI) and a CVC lumen thrombosis among the most serious. Standard of care regarding CVCs includes their sealing with heparin lock solutions to prevent catheter lumen thrombosis. Other lock solutions to prevent CRI, such as antimicrobial lock solutions, have proven useful with antibiotics solutions, but not as yet for non-antibiotic antimicrobial solutions. Furthermore, it is uncertain if these solutions have a negative effect on thrombosis incidence. OBJECTIVES: To assess the efficacy and safety of antimicrobial (antibiotic, non-antibiotic, or both) catheter lock solutions for preventing CRI in participants undergoing HD with a CVC. SEARCH METHODS: We searched the Cochrane Kidney and Transplant Specialised Register up to 18 December 2017 through contact with the Information Specialist using search terms relevant to this review. Studies in the Register are identified through searches of CENTRAL, MEDLINE, and EMBASE, conference proceedings, the International Clinical Trials Register (ICTRP) Search Portal, and ClinicalTrials.gov. SELECTION CRITERIA: We included all randomised or quasi-randomised control trials (RCTs) comparing antimicrobial (antibiotic and non-antibiotic) lock solutions to standard lock solutions, in participants using a CVC for HD, without language restriction. DATA COLLECTION AND ANALYSIS: Two authors independently assessed studies for eligibility, and two additional authors assessed for risk of bias and extracted data. We expressed results as rate ratios (RR) per 1000 catheter-days or 1000 dialysis sessions with 95% confidence intervals (CI). Statistical analyses were performed using the random-effects model. MAIN RESULTS: Thirty-nine studies, enrolling 4216 participants, were included in this review, however only 30 studies, involving 3392 participants, contained enough data to be meta-analysed. Risk of bias was low or unclear for most domains in the majority of the included studies.Studies compared antimicrobial lock solutions (antibiotic and non-antibiotic) to standard sealing solutions (usually heparin) of the CVC for HD. Fifteen studies used antibiotic lock solutions, 21 used non-antibiotic antimicrobial lock solutions, and 4 used both (antibiotic and non-antibiotic) lock solutions. Studies reported the incidence of CRI, catheter thrombosis, or both.Antimicrobial lock solutions probably reduces CRI per 1000 catheter-days (27 studies: RR 0.38, 95% CI 0.27 to 0.53; I2 = 54%; low certainty evidence), however antimicrobial lock solutions probably makes little or no difference to the risk of thrombosis per 1000 catheter days (14 studies: RR 0.79, 95% CI 0.52 to 1.22; I2 = 83%; very low certainty evidence). Subgroup analysis of antibiotic and the combination of both lock solutions showed that both probably reduced CRI per 1000 catheter-days (13 studies: RR 0.30, 95% CI: 0.22 to 0.42; I2 = 47%) and risk of thrombosis per 1000 catheter-days (4 studies: RR 0.26, 95% CI: 0.14 to 0.49; I2 = 0%), respectively. Non-antibiotic antimicrobial lock solutions probably reduced CRI per 1000 catheter-days for tunnelled CVC (9 studies: RR 0.60, 95% CI 0.40 to 0.91) but probably made little or no difference with non-tunnelled CVC (4 studies: RR 0.93, 95% CI 0.48 to 1.81). Subgroup analyses showed that antibiotic (5 studies: RR 0.76, 95% CI 0.42 to 1.38), non-antibiotic (8 studies: RR 0.85, 95% CI 0.44 to 1.66), and the combination of both lock solutions (3 studies: RR 0.63, 95% CI 0.22 to 1.81) made little or no difference to thrombosis per 1000 catheter-days compared to control lock solutions. AUTHORS' CONCLUSIONS: Antibiotic antimicrobial and combined (antibiotic-non antibiotic) lock solutions decreased the incidence of CRI compared to control lock solutions, whereas non-antibiotic lock solutions reduce CRI only for tunnelled CVC. The effect on thrombosis incidence is uncertain for all antimicrobial lock solutions. Our confidence in the evidence is low and very low; therefore, better-designed studies are needed to confirm the efficacy and safety of antimicrobial lock solutions.

Surface plasmon resonance imaging (SPRi) for analysis of DNA aptamer:ß-conglutin interactions.

Surface plasmon resonance imaging (SPRi) is a label-free detection method that offers a suitable and reliable platform for the real time monitoring of biomolecular interactions. In the work reported here, SPRi was used to evaluate the affinity and specificity of three different aptamers selected against the Lup an 1 anaphylactic allergen ß-conglutin (ß-conglutin binding aptamers I and II (ß-CBA I and ß-CBA II)), as well as an 11-mer truncated version of ß-CBA I. Thiol modified aptamers were immobilised on a gold substrate through a self-assembling process and the use of different blocking strategies to prevent non-specific binding were evaluated. Dissociation constants of 20, 13 and 1 nM were determined for ß-CBA I, ß-CBA II and the 11-mer truncated aptamer, respectively. The three aptamers were then studied in various different sandwich formats and the ß-CBA I/11-mer and ß-CBA II were observed to bind to different aptatopes on the target protein. Each of the aptamers were then used either as surface immobilised aptamer, or as reporter aptamer, and added with the protein target ß-conglutin in either a sequential of simultaneous manner, and the changes in SPR signal monitored. The preferred approach for formation of a sandwich aptacomplex was with immobilised ß-CBA II, followed by addition of pre-incubated ß-conglutin and 11-mer, whilst addition of the 11-mer following addition of the ß-conglutin, resulted in displacement of the bound target. The ability to provide parallel qualitative and quantitative detection establishes SPRi as a powerful tool for the study of immobilised aptamer-target interactions.

Ultrasensitive and rapid detection of ß-conglutin combining aptamers and isothermal recombinase polymerase amplification.

Lupin is increasingly being used in a variety of food products due to its nutritional, functional and nutraceutical properties. However, several examples of severe and even fatal food-associated anaphylaxis due to lupin inhalation or ingestion have been reported, resulting in the lupin subunit ß-conglutin, being defined as the Lup an 1 allergen by the International Union of Immunological Societies (IUIS) in 2008. Here, we report an innovative method termed aptamer-recombinase polymerase amplification (Apta-RPA) exploiting the affinity and specificity of a DNA aptamer selected against the anaphylactic ß-conglutin allergen termed ß-conglutin binding aptamer II (ß-CBA II), facilitating ultrasensitive detection via isothermal amplification. Combining magnetic beads as the solid phase with Apta-RPA detection, the total assay time was reduced from 210 min to just 25 min, with a limit of detection of 3.5 × 10-11 M, demonstrating a rapid and ultrasensitive generic methodology that can be used with any aptamer. Future work will focus on further simplification of the assay to a lateral flow format. Graphical Abstract Schematic representation of the rapid and novel bead-based Apta-RPA assay.

Aptamer Lateral Flow Assays for Ultrasensitive Detection of ß-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers.

In this work, different methodologies were evaluated in search of robust, simple, rapid, ultrasensitive, and user-friendly lateral flow aptamer assays. In one approach, we developed a competitive based lateral flow aptamer assay, in which ß-conglutin immobilized on the test line of a nitrocellulose membrane and ß-conglutin in the test sample compete for binding to AuNP labeled aptamer. The control line exploits an immobilized DNA probe complementary to the labeled aptamer, forcing displacement of the aptamer from the ß-conglutin-aptamer complex. In a second approach, the competition for aptamer binding takes place off-strip, and following competition, aptamer bound to the immobilized ß-conglutin is eluted and used as a template for isothermal recombinase polymerase amplification, exploiting tailed primers, resulting in an amplicon of a duplex flanked by single stranded DNA tails. The amplicon is rapidly and quantitatively detected using a nucleic acid lateral flow with an immobilized capture probe and a gold nanoparticle labeled reporter probe. The competitive lateral flow is completed in just 5 min, achieving a detection limit of 55 pM (1.1 fmol), and the combined competitive-amplification lateral flow requires just 30 min, with a detection limit of 9 fM (0.17 amol).

ß-Conglutin dual aptamers binding distinct aptatopes.

An aptamer was previously selected against the anaphylactic allergen ß-conglutin (ß-CBA I), which was subsequently truncated to an 11-mer and the affinity improved by two orders of magnitude. The work reported here details the selection and characterisation of a second aptamer (ß-CBA II) selected against a second aptatope on the ß-conglutin target. The affinity of this second aptamer was similar to that of the 11-mer, and its affinity was confirmed by three different techniques at three independent laboratories. This ß-CBA II aptamer in combination with the previously selected ß-CBA I was then exploited to a dual-aptamer approach. The specific and simultaneous binding of the dual aptamer (ß-CBA I and ß-CBA II) to different sites of ß-conglutin was confirmed using both microscale thermophoresis and surface plasmon resonance where ß-CBA II serves as the primary capturing aptamer and ß-CBA I or the truncated ß-CBA I (11-mer) as the secondary signalling aptamer, which can be further exploited in enzyme-linked aptamer assays and aptasensors.

[Recommendations of health care users and professionals to achieve a timely access to HIV diagnosis]. / Optimizando el acceso oportuno al test de ELISA para el diagnóstico del VIH: Recomendaciones desde los usuarios y profesionales de la Atención Primaria de Salud.

BACKGROUND: Early HIV (human immunodeficiency virus) diagnosis optimizes therapies aimed at reducing viral load, increasing survival, lowering health costs and reducing the number of people infected with the virus. In Chile, despite widespread and readily available HIV testing, infected people continue to get tested in a late fashion and are usually diagnosed in advanced stages of the disease. AIM: To determine the elements that facilitate or impede a timely HIV testing and to evaluate how to improve the access to HIV testing. MATERIAL AND METHODS: Descriptive, in-depth interviews to 30 participants with unknown serology, 15 participants diagnosed at AIDS stage and 15 health care professionals working at a primary healthcare settings. RESULTS: Users and professionals formulated three suggestions to improve timely access to ELISA test for HIV diagnosis. Namely, to inform users and professionals about the characteristics of the disease and diagnostic test, to offer fast and easy access to HIV testing, and to train the whole healthcare team about obtaining informed consent for testing. CONCLUSIONS: These recommendations should be implemented at healthcare centers to attain a timely HIV diagnosis.

[Facilitators and barriers to HIV testing: a literature review]. / Facilitadores y barreras que enfrentan las personas al tomarse el test de ELISA para el diagnóstico del VIH: revisión de la literatura.

BACKGROUND: Late diagnosis of HIV is a problem of international and national relevance. Despite the availability of HIV testing in primary health care, it is often performed too late. AIM: To identify facilitators and barriers to early HIV testing in primary health care. METHODS: Four databases of nursing, psychological, biomedical, and health related professions areas were examined with a review protocol. Results were grouped into two main subjects: facilitators and barriers occurring among the population, among health care workers, and within primary health care centers. RESULTS: Perception of risk behaviors, self-care, social support, trust, confidentiality of the examination, the offer of the examination, and the knowledge of early treatment have been recognized as facilitators for taking the exam. The lack of information about the test and the disease are recognized as the main barrier to access the test. This information is a cornerstone to design and implement strategies to increase the number of people taking voluntarily HIV testing.

People With Diabetes and Caregivers Prefer Rescue Glucagon Treatment With a Wider Storage Temperature Range and a Nasal Administration, When Efficacy is Similar: A Discrete Choice Experiment in Spain.

BACKGROUND: Conventional injectable glucagon (IG) and nasal glucagon (NG), both having similar efficacy, are two options for the emergency treatment of severe hypoglycemia in Spain. This study elicited the effect of changes in key attributes on preferences for NG and IG medication profiles of people with diabetes and caregivers in Spain. METHODS: The relative attribute importance (RAI) that participants placed on glucagon preparation, preparation and administration time, delivery method, recovery time, device size, storage temperature, and headache risk was estimated from an online discrete choice experiment. In addition, patients and caregivers were presented with NG and IG profiles that included rates of successful administration; the proportion of participants choosing each profile was summarized. RESULTS: The analysis included 276 adults with diabetes (65% type 1) and 270 caregivers (49% type 1). Overall mean age was 40 years; 51% were female. The most important attributes were storage temperature (RAI [95% confidence interval] = 27.3% [22.9-32.2]) and delivery method (17.4% [13.1-21.9]). Headache risk (16.2% [11.8-20.7]), time to prepare and administer (14.5% [10.1-18.8]), glucagon preparation (11.4% [6.8-15.8]), recovery time (8.9% [4.3-13.3]), and device size (4.3% [0.3-8.8]) were also relevant. When comparing medication profiles, significantly more participants (78%) preferred NG over IG profiles (P < .001). CONCLUSION: Adults with diabetes and caregivers prefer a glucagon treatment with a higher rate of successful administration, wider storage temperature, and nasal delivery method, when efficacy is similar. Participants favored NG over conventional IG as a rescue medication for severe hypoglycemia. This information may help decision-making by payers and treatment discussions between health care professionals and patients.

Ultrasensitive determination of ß-conglutin food allergen by means an aptamer assay based on inductively coupled plasma mass spectrometry detection.

The overall objective of this work is the evaluation of different competitive aptamer assays based on inductively coupled plasma mass spectrometry (ICP-MS) detection for the determination of ß-conglutin (food protein allergen from lupin) in flour samples. To this end, two competitive aptamer assay schemes were developed using either thiolated aptamers chemisorbed onto gold nanoparticles (AuNPs) or biotinylated aptamers linked to streptavidin-AuNPs. The influence of ICP-MS detection mode (i.e., conventional vs single particle) on assay performance was explored. In the case of the thiolated aptamer, the limit of detection (LoD) obtained using the single particle mode was improved 2-fold as compared to the LoD provided by the conventional mode. With regards to the biotinylated aptamer, the use of the conventional mode provided a 5-fold improvement of LoD as compared to that obtained for the single particle one. Using the optimized conditions, the best LoD of 2 pM was obtained with the biotinylated aptamer operating with conventional ICP-MS detection. When compared to previous reports using the same aptamer in a competitive assay, the developed method significantly improved the LoD by at least an order of magnitude. Different flour samples containing lupin were successfully analyzed according to European Conformity guidelines for the analysis of food contaminants.

Generic Platform for the Multiplexed Targeted Electrochemical Detection of Osteoporosis-Associated Single Nucleotide Polymorphisms Using Recombinase Polymerase Solid-Phase Primer Elongation and Ferrocene-Modified Nucleoside Triphosphates.

Osteoporosis is a multifactorial disease influenced by genetic and environmental factors, which contributes to an increased risk of bone fracture, but early diagnosis of this disease cannot be achieved using current techniques. We describe a generic platform for the targeted electrochemical genotyping of SNPs identified by genome-wide association studies to be associated with a genetic predisposition to osteoporosis. The platform exploits isothermal solid-phase primer elongation with ferrocene-labeled nucleoside triphosphates. Thiolated reverse primers designed for each SNP were immobilized on individual gold electrodes of an array. These primers are designed to hybridize to the SNP site at their 3'OH terminal, and primer elongation occurs only where there is 100% complementarity, facilitating the identification and heterozygosity of each SNP under interrogation. The platform was applied to real blood samples, which were thermally lysed and directly used without the need for DNA extraction or purification. The results were validated using Taqman SNP genotyping assays and Sanger sequencing. The assay is complete in just 15 min with a total cost of 0.3€ per electrode. The platform is completely generic and has immense potential for deployment at the point of need in an automated device for targeted SNP genotyping with the only required end-user intervention being sample addition.

Exploiting the Nucleic Acid Nature of Aptamers for Signal Amplification.

Aptamer-based assays and sensors are garnering increasing interest as alternatives to antibodies, particularly due to their increased flexibility for implementation in alternative assay formats, as they can be employed in assays designed for nucleic acids, such as molecular aptamer beacons or aptamer detection combined with amplification. In this work, we took advantage of the inherent nucleic acid nature of aptamers to enhance sensitivity in a rapid and facile assay format. An aptamer selected against the anaphylactic allergen ß-conglutin was used to demonstrate the proof of concept. The aptamer was generated by using biotinylated dUTPs, and the affinity of the modified aptamer as compared to the unmodified aptamer was determined by using surface plasmon resonance to calculate the dissociation constant (KD), and no significant improvement in affinity due to the incorporation of the hydrophobic biotin was observed. The modified aptamer was then applied in a colorimetric competitive enzyme-linked oligonucleotide assay, where ß-conglutin was immobilized on the wells of a microtiter plate, competing with ß-conglutin free in solution for the binding to the aptamer. The limit of detection achieved was 68 pM, demonstrating an improvement in detection limit of three orders of magnitude as compared with the aptamer simply modified with a terminal biotin label. The concept was then exploited by using electrochemical detection and screen-printed electrodes where detection limits of 326 fM and 7.89 fM were obtained with carbon and gold electrodes, respectively. The assay format is generic in nature and can be applied to all aptamers, facilitating an easy and cost-effective means to achieve lower detection limits.