A Genetic Lab-on-Phone Test for Point-of-Care Diagnostic of Lactose Intolerance near Patient and in less than 90 Minutes.

Background: The -13910 C/T single nucleotide polymorphism located within the MCM6 gene, an enhancer region located upstream of the lactase-phlorizin hydrolase gene, is associated with lactase persistence/non-persistence traits among the Caucasian population. The performance of a new point-of-care CE-IVD (In Vitro Diagnostic) marked isothermal lab-on-phone lactose intolerance assay, using crude samples, was assessed in comparison with Sanger sequencing using purified DNA, as reference method.

Methods: The study was conducted following a non-probability sampling using direct buccal swab (n = 63) and capillary blood (n = 43) clinical samples from a total of 63 volunteers.

An isothermal lab-on-phone test for easy molecular diagnosis of SARS-CoV-2 near patients and in less than 1 hour.

Objectives: The performance of a new point-of-care CE-IVD-marked isothermal lab-on-phone COVID-19 assay was assessed in comparison to a gold standard real-time reverse transcriptase-PCR method.

Methods: The study was conducted following a nonprobability sampling of ≥16-year-old volunteers from three different laboratories, using direct mouthwash (N = 24) or nasopharyngeal (N = 191) clinical samples.

Results: The assay demonstrated 95.

Epitope Mapping by NMR of a Novel Anti-Aβ Antibody (STAB-MAb).

Alzheimer´s Disease (AD) is one of the most common neurodegenerative disorders worldwide. Excess of β-amyloid (Aβ), a peptide with a high propensity to misfold and self-aggregate, is believed to be the major contributor to the observed neuronal degeneration and cognitive decline in AD. Here, we characterize the epitope of a novel anti-Aβ monoclonal antibody, the STAB-MAb, which has previously demonstrated picomolar affinities for both monomers (K = 80 pM) and fibrils (K = 130 pM) of Aβ(1-42) and has shown therapeutic efficacy in preclinical mouse models of AD.

Intranasal insulin activates Akt2 signaling pathway in the hippocampus of wild-type but not in APP/PS1 Alzheimer model mice.

Type 2 diabetes mellitus (T2DM) increases the risk for Alzheimer's disease (AD). Human AD brains show reduced glucose metabolism as measured by [18F]fluoro-2-deoxy-2-D-glucose positron emission tomography (FDG-PET). Here, we used 14-month-old wild-type (WT) and APP/PS1 (APP/PS1) transgenic mice to investigate how a single dose of intranasal insulin modulates brain glucose metabolism using FDG-PET and affects spatial learning and memory.

Allele specific LAMP- gold nanoparticle for characterization of single nucleotide polymorphisms.

Due to their relevance as disease biomarkers and for diagnostics, screening of single nucleotide polymorphism (SNPs) requires simple and straightforward strategies capable to provide results in medium throughput settings. Suitable approaches relying on isothermal amplification techniques have been evolving to substitute the cumbersome and highly specialized PCR amplification detection schemes. Nonetheless, identification of an individual's genotype still requires sophisticated equipment and laborious methods.

Characterization of genomic single nucleotide polymorphism via colorimetric detection using a single gold nanoprobe.

Identification of specific nucleic acid sequences mediated by gold nanoparticles derivatized thiol-modified oligonucleotides (Au-nanoprobes) has been proven to be a useful tool in molecular diagnostics. Here, we demonstrate that, on optimization, detection may be simplified via the use of a single Au-nanoprobe to detect a single nucleotide polymorphism (SNP) in homo- or heterozygote condition. We validated this non-cross-linking approach through the analysis of 20 clinical samples using a single specific Au-nanoprobe for an SNP in the FTO (fat mass and obesity-associated) gene against direct DNA sequencing.

Association of FTO and PPARG polymorphisms with obesity in Portuguese women.

Purpose: We evaluated the association between risk of obesity in the Portuguese population and two obesity-related single-nucleotide gene polymorphisms: fat-mass and obesity-associated (FTO) rs9939609 and peroxisome proliferator-activated receptor gamma (PPARG) rs1801282.

Patients And Methods: A total of 194 Portuguese premenopausal female Caucasians aged between 18 and 50 years (95 with body mass index [BMI] ≥30 g/m(2), 99 controls with BMI 18.5-24.

RNA quantification using noble metal nanoprobes: simultaneous identification of several different mRNA targets using color multiplexing and application to cancer diagnostics.

Nanotechnology provides new tools for gene expression analysis that allow for sensitive and specific characterization of prognostic signatures related to cancer. Cancer is a multigenic complex disease where multiple gene loci contribute to the phenotype. The ability to simultaneously monitor differential expression originating from each locus allows for a more accurate indication of degree of cancerous activity than either locus alone.

Noble metal nanoparticles for biosensing applications.

In the last decade the use of nanomaterials has been having a great impact in biosensing. In particular, the unique properties of noble metal nanoparticles have allowed for the development of new biosensing platforms with enhanced capabilities in the specific detection of bioanalytes. Noble metal nanoparticles show unique physicochemical properties (such as ease of functionalization via simple chemistry and high surface-to-volume ratios) that allied with their unique spectral and optical properties have prompted the development of a plethora of biosensing platforms.

Nanoparticles in molecular diagnostics.

The aim of this chapter is to provide an overview of the available and emerging molecular diagnostic methods that take advantage of the unique nanoscale properties of nanoparticles (NPs) to increase the sensitivity, detection capabilities, ease of operation, and portability of the biodetection assemblies. The focus will be on noble metal NPs, especially gold NPs, fluorescent NPs, especially quantum dots, and magnetic NPs, the three main players in the development of probes for biological sensing. The chapter is divided into four sections: a first section covering the unique physicochemical properties of NPs of relevance for their utilization in molecular diagnostics; the second section dedicated to applications of NPs in molecular diagnostics by nucleic acid detection; and the third section with major applications of NPs in the area of immunoassays.

Gold nanoparticle-based fluorescence immunoassay for malaria antigen detection.

The development of rapid detection assays for malaria diagnostics is an area of intensive research, as the traditional microscopic analysis of blood smears is cumbersome and requires skilled personnel. Here, we describe a simple and sensitive immunoassay that successfully detects malaria antigens in infected blood cultures. This homogeneous assay is based on the fluorescence quenching of cyanine 3B (Cy3B)-labeled recombinant Plasmodium falciparum heat shock protein 70 (PfHsp70) upon binding to gold nanoparticles (AuNPs) functionalized with an anti-Hsp70 monoclonal antibody.

Noble metal nanoparticles applications in cancer.

Nanotechnology has prompted new and improved materials for biomedical applications with particular emphasis in therapy and diagnostics. Special interest has been directed at providing enhanced molecular therapeutics for cancer, where conventional approaches do not effectively differentiate between cancerous and normal cells; that is, they lack specificity. This normally causes systemic toxicity and severe and adverse side effects with concomitant loss of quality of life.

Portable optoelectronic biosensing platform for identification of mycobacteria from the Mycobacterium tuberculosis complex.

In this paper we report on the fabrication and performance of a portable and low cost optoelectronic platform integrating a double color tuned light emitting diode as light source, an amorphous/nanocrystalline silicon photodetector with a flat spectral response in the wavelength range from 520 nm to 630 nm and integrated electronic for signal acquisition and conditioning constituted by current to voltage converter, a filter and an amplification stage, followed by an analog to digital converter, with appropriate software for full automation to minimize human error. Incorporation of the double color tuned light emitting diode provides for a simple yet innovative solution to signal acquisition independently from the light intensity and/or solution concentration, while considerably decreasing production costs. Detection based on Au-nanoprobes constitutes the biorecognition step and allowed identification of specific sequences of Mycobacterium tuberculosis complex, namely Mycobacterium bovis and M.

Development of a fast and efficient ultrasonic-based strategy for DNA fragmentation.

Several ultrasound-based platforms for DNA sample preparation were evaluated in terms of effective fragmentation of DNA (plasmid and genomic DNA)-ultrasonic probe, sonoreactor, ultrasonic bath and the newest Vialtweeter device. The sonoreactor showed the best efficiency of DNA fragmentation while simultaneously assuring no cross-contamination of samples, and was considered the best ultrasonic tool to achieve effective fragmentation of DNA at high-throughput and avoid sample overheating. Several operation variables were studied-ultrasonication time and amplitude, DNA concentration, sample volume and sample pre-treatment-that allowed optimisation of a sonoreactor-based strategy for effective DNA fragmentation.

Optimizing Au-nanoprobes for specific sequence discrimination.

Gold nanoparticles functionalized with thiol-oligonucleotides are ideal platforms for detection of specific DNA sequences. Here we evaluate the effect of single base mismatches in hybridization efficiency according to the position of the mismatch, base pairing combination and thiol-oligonucleotide density in terms of specificity and efficiency of target recognition. Hybridization efficiency and single-nucleotide polymorphism discrimination at room temperature is maximized at a density of 83+/-4 thiol-oligonucleotides per 13.

Inkjet printed and "doctor blade" TiO2 photodetectors for DNA biosensors.

A dye sensitized TiO(2) photodetector has been integrated with a DNA detection method based on non-cross-linking hybridization of DNA-functionalized gold nanoparticles, resulting in a disposable colorimetric biosensor. We present a new approach for the fabrication of dye sensitized TiO(2) photodetectors by an inkjet printing technique-a non-contact digital, additive, no mask and no vacuum patterning method, ideal for cost efficient mass production. The developed biosensor was compared against a dye sensitized photodetector fabricated by the traditional "doctor blade" method.

Imaging gold nanoparticles for DNA sequence recognition in biomedical applications.

The hybridization of single-stranded oligonucleotide-derivatized gold nanoparticles (Au nanoprobes) with double stranded complementary DNA was directly observed by atomic force microscopy (AFM). This specific interaction is the basis for an Au nanoprobe-based homogeneous assay for specific DNA sequence detection, based on salt-induced particle aggregation that is prevented when a complementary target is present. For long DNA targets (linearized plasmid DNA) complicated hybridized target DNA-Au-nanoprobes structures were formed, that were interpreted as the basis for stability of the Au nanoprobes against salt-induced aggregation.

Gold nanoparticles for the development of clinical diagnosis methods.

The impact of advances in nanotechnology is particularly relevant in biodiagnostics, where nanoparticle-based assays have been developed for specific detection of bioanalytes of clinical interest. Gold nanoparticles show easily tuned physical properties, including unique optical properties, robustness, and high surface areas, making them ideal candidates for developing biomarker platforms. Modulation of these physicochemical properties can be easily achieved by adequate synthetic strategies and give gold nanoparticles advantages over conventional detection methods currently used in clinical diagnostics.

Colorimetric detection of eukaryotic gene expression with DNA-derivatized gold nanoparticles.

Thiol-linked DNA-gold nanoparticles were used in a novel colorimetric method to detect the presence of specific mRNA from a total RNA extract of yeast cells. The method allowed detection of expression of the FSY1 gene that encodes a specific fructose/H+ symporter in Saccharomyces bayanus PYCC 4565. FSY1 is strongly expressed when the yeast is grown in fructose as the sole carbon source, while cells cultivated in glucose as the sole carbon source repress gene expression.