Nucleic Acid Hybridization Assay Research Articles

Developmental PFOS exposure alters lung inflammation and barrier integrity in juvenile mice.

Emerging epidemiological evidence indicates perfluorooctane sulfonic acid (PFOS) is increasingly associated with asthma and respiratory viral infections. Animal studies suggest PFOS disrupts lung development and immuno-inflammatory responses, but little is known about the potential consequences on respiratory health and disease risk. Importantly, PFOS exposure during the critical stages of lung development may increase disease risk later in life. Thus, we hypothesized that developmental PFOS exposure will affect lung inflammation and alveolar/airway development in a sex-dependent manner. To address this knowledge gap, timed pregnant Balb/cJ dams were orally dosed with a PFOS (1.0 or 2.0 mg/kg/d) injected mealworm or a vehicle control daily from gestational day (GD) 0.5 to postnatal day (PND) 21, and offspring were sacrificed at PND 22-23. PFOS-exposed male offspring displayed increased alveolar septa thickness. Occludin was also downregulated in the lungs after PFOS exposure in mice, indicative of barrier dysfunction. BALF macrophages were significantly elevated at 2.0 mg/kg/d PFOS in both sexes compared with vehicles, whereas BALF cytokines (TNF-α, IL-6, KC, MIP-1α, MIP-1β, and MCP-1) were suppressed in PFOS-exposed male offspring compared with vehicle controls. Multiplex nucleic acid hybridization assay showed male-specific downregulation of cytokine gene expression in PFOS-exposed mice compared with vehicle mice. Overall, these results demonstrate PFOS exposure exhibits male-specific adverse effects on lung development and inflammation in juvenile offspring, possibly predisposing them to later-in-life respiratory disease. Further research is required to elucidate the mechanisms underlying the sex-differentiated pulmonary toxicity of PFOS.

Abstract C136: Magnetic nanoparticles and quantum dots coupled visual confirmation of cervical cancer cells from cytology samples: A cost-effective, quick and suitable test for rural Indian set-up

Abstract Introduction Screening and management of cervical cancer is a National health priority of developed and developing countries like India. Cervical cancer screening and diagnosis are currently done by cervical pap screening, colposcopy, and nucleic acid hybridization and amplification assays, which are time-consuming and require specialized facilities. These shortcomings pose severe restrictions on the feasibility of the techniques for a country like India.Aim and objectives Bearing this in mind, we aimed to develop a simple, cost-effective, and visual detection of cervical cancer, based on magnetic nanoparticles coupled quantum dots immuno-nano-fluorescence assay (MNCQDINFA).Materials and methods The detection system comprised of magnetic nanoparticles(MNP) and quantum dots (QD) conjugated specific polyclonal antibodies complex generated against recombinantly purified individual domains (domainN and domainC) of HPV-16 oncoprotein E7, which is deregulated and overexpressed to detectable limits by transformed cervical cancer cells only [1-3]. The assay procedure involves incubating processed cervical cytology samples with MNP-Ab (domainN) complex, magnet-induced pelleting, incubation with QD-Ab (domainC) complex, magnet-assisted washing, and final reconstitution in sample buffer and visualization under UV (Figure). Reconstituted sample in the presence of UV gives fluorescence as an indication of the presence of E7 protein thus cervical cancer cells. Results This method produces a visual fluorescence exclusively in the presence of transformed cervical cancer cells under UV light. Analytical assays showed that a minimum of 0.1µg of E7 protein could be detected specifically from a pool of cellular proteins. HPV 16-positive cervical cancer cell lysates showed concordant results. Both the diagnostic sensitivity and specificity of the assay were determined to be 100%. Conclusion The proposed invention is a simple, visual (naked eye), quick (4 hours), and cost-effective (INR 200-300) cervical cancer cells detection method necessary to identify the candidates at risk so that suitable and timely intervention can be provided.

Application of Fluorescent In Situ Hybridization for Quick Identification of Microorganisms from Positive Blood Cultures

AIM: The aim of this study was to evaluate the diagnostic potential of the fluorescent in situ hybridization (FISH) method for quick identification of microorganisms from positive blood cultures. MATERIALS AND METHODS: QuickFISH BC is a multicolor, qualitative nucleic acid hybridization assay using specific fluorescent-labeled probes for identification of Gram-positive bacteria (S. aureus, Coagulase-negative Staphylococcus spp. – CoNS, E. faecalis, and E. faecium); Gram-negative bacteria (E. coli, P. aeruginosa, and K. pneumoniaе), and fungi (C. albicans, C. tropicalis, and C. glabrata). This method applied to 72 positive blood cultures obtained from patients admitted at the University Hospital St. George – Plovdiv. A preliminary selection based on Gram staining was performed before the application of the FISH test. All microorganisms were subject to identification by routine biochemical tests, semi-automated and automated systems as well. Statistical data processing included descriptive statistics, nonparametric analysis for testing hypotheses by SPSS v. 22.0, and Microsoft Excel software. p < 0.05 was considered statistically significant. RESULTS: FISH detected microorganisms in 63 (87.5%) positive blood cultures, whereas no fluorescent signal was observed in 9 (12.5%). The latter was because not all the microorganisms we identified are included in the test spectrum, for example – Enterobacter spp. and Acinetobacter spp. By FISH, we found S. aureus in 10 (15.9%) cases, CoNS in 20 (31.6%), E. faecalis in 4 (6.4%), and E. faecium in 4 (6.4%). E. coli (n = 7; 11.1%) was the leading cause of bacteremia among Gram-negative bacteria, whereas C. albicans predominated (n = 4; 6.4%) among fungi. CONCLUSION: QuickFISH BC is a rapid and accurate screening method for the identification of some of the most frequent pathogens causing bacteremia. This enables the initiation of the early and adequate antimicrobial therapy. The lack of pathogen identification from positive blood cultures using this method implies the need to continue identification with other tests.

Split-type electrochemiluminescent gene assay platform based on gold nanocluster probe for human papillomavirus diagnosis

Persistent high-risk human papillomavirus (HPV) infection is the leading cause of cervical cancer. Efficient detection of HPV16 E7 is necessary for early diagnosis and cure of the disease. Here, a novel and high-performance Au nanocluster (AuNC) probe-based split-type electrochemiluminescent (ECL) assay platform has been established to detect these oncogenes, in which the nucleic acid hybridization assay and the ECL measurements are performed independently. The proposed approach combines superior magnetic nanobead enrichment and separation technology, specific nucleic acid hybridization technology, and high-efficiency AuNC probe ECL strategy, and shows excellent advantages. First, the split-type ECL sensing platform can effectively avoid interference from biological samples and adequately uses the ECL efficiency of the AuNC probe. Furthermore, the ultrahigh sensitivity assay of HPV DNA can be achieved without any complex nucleic acid amplification technique. Taking advantage of the above merits of split-type detection, the ECL DNA sensor achieved ideal low detection of 6.8 aM and a wide dynamic range bridging 10 orders of magnitude HPV16 E7. Furthermore, together with its favorable and powerful specificity, high sensitivity, and good selectivity, this strategy could detect HPV16 E7 DNA in human samples, which showed great consistency with the FDA-approved approach (Hybrid capture 2, HC2). Therefore, this work proposes a facile and reliable split-type ECL platform for HPV diagnosis and shows great potential for the early diagnosis of other diseases.

Simultaneous detection of small molecules, proteins and microRNAs using single molecule arrays.

Biological samples such as blood, urine, cerebrospinal fluid and saliva contain a large variety of proteins, nucleic acids, and small molecules. These molecules can serve as potential biomarkers of disease and therefore, it is desirable to simultaneously detect multiple biomarkers in one sample. Current detection techniques suffer from various limitations including low analytical sensitivity and complex sample processing. In this work, we present an ultrasensitive method for simultaneous detection of small molecules, proteins and microRNAs using single molecule arrays (Simoa). Dye-encoded beads modified with specific capture probes were used to quantify each analyte. Multiplex competitive Simoa assays were established for simultaneous detection of cortisol and prostaglandin E2. In addition, competitive and sandwich immunoassays were combined with a direct nucleic acid hybridization assay for simultaneous detection of cortisol, interleukin 6 and microRNA 141. The multi-analyte Simoa assay shows high sensitivity and specificity, which provides a powerful tool for the analysis of many different samples.

Nonfaradaic Current Suppression in DNA-Based Electrochemical Assays with a Differential Potentiostat.

One of the key factors limiting sensitivity in many electrochemical assays is the nonfaradaic or capacitive current. This is particularly true in modern assay systems based on DNA monolayers at gold electrode surfaces, which have shown great promise for bioanalysis in complex milieu such as whole blood or serum. While various changes in analytical parameters, redox reporter molecules, DNA structures, probe coverage, and electrode surface area have been shown useful, background reduction by hardware subtraction has not yet been explored for these assays. Here, we introduce new electrochemistry hardware that considerably suppresses nonfaradaic currents through real-time analog subtraction during current-to-voltage conversion in the potentiostat. This differential potentiostat (DiffStat) configuration is shown to suppress or remove capacitance currents in chronoamperometry, cyclic voltammetry, and square-wave voltammetry measurements applied to nucleic acid hybridization assays at the electrode surface. The DiffStat makes larger electrodes and higher sensitivity settings accessible to the user, providing order-of-magnitude improvements in sensitivity, and it also significantly simplifies data processing to extract faradaic currents in square-wave voltammetry (SWV). Because two working electrodes are used for differential measurements, unique arrangements are introduced such as converting signal-OFF assays to signal-ON assays or background drift correction in 50% human serum. Overall, this new potentiostat design should be helpful not only in improving the sensitivity of most electrochemical assays, but it should also better support adaptation of assays to the point-of-care by circumventing complex data processing.

Prevalence of oral HPV infection in cervical HPV positive women and their sexual partners.

Human papillomavirus (HPV) infection represents the primary cause of anogenital premalignant and malignant disease. Regarding the high prevalence of cervical HPV infection and the increasing incidence of HPV associated oropharyngeal cancer in recent years, a significant viral transmission from the cervical to the oral site, possibly depending on the sexual behavior must be considered. The present study aims to determine the prevalence of oral HPV infection in cervical HPV positive and negative women and their sexual partners. Cervical HPV positive and negative women and their sexual partners took part in the study. Cervical smears, oral smears and mouthwashes were taken from women attending gynecological outpatient clinics in two different institutions. Further, oral smears as well as mouthwashes of their sexual partners were obtained whenever possible. HPV genotyping was performed using the Cobas® polymerase chain reaction and nucleic acid hybridization assay for the detection of 14 high-risk HPV types. In addition, all participants were invited to complete a personal questionnaire. 144 HPV positive and 77 HPV negative women and altogether 157 sexual partners took part in the study. Age, sexual behaviour, medication, smoking and alcohol consumption were distributed equally in both groups. Cervical HPV positive women had a significantly higher number of sexual partners. One woman with a HPV positive cervical smear and one partner of a woman with a HPV positive cervical smear showed an oral HPV infection. No oral HPV infections were detected in the HPV negative control group. The overall incidence of oral HPV infection was 0.5%, the incidence of oral HPV infection in women with a positive cervical smear was 0.7%. The data demonstrate that the overall risk of an oral HPV infection is low. HPV transmission to the oropharynx by autoinoculation or oral-genital contact constitute a rare and unlikely event.

Detection of cystic fibrosis transmembrane conductance regulator ΔF508 gene mutation using a paper-based nucleic acid hybridization assay and a smartphone camera.

Diagnostic technology that makes use of paper platforms in conjunction with the ubiquitous availability of digital cameras in cellular telephones and personal assistive devices offers opportunities for development of bioassays that are cost effective and widely distributed. Assays that operate effectively in aqueous solution require further development for implementation in paper substrates, overcoming issues associated with surface interactions on a matrix that offers a large surface-to-volume ratio and constraints on convective mixing. This report presents and compares two related methods for determination of oligonucleotides that serve as indicators of cystic fibrosis, differentiating between the normal wild-type sequence, and a mutant-type sequence that has a 3-base replacement. The transduction strategy operates by selective hybridization of oligonucleotide probes that are conjugated to fluorescent quantum dots, where hybridization of target sequences causes a molecular fluorophore to approach the quantum dot and become emissive through fluorescence resonance energy transfer. Detection can rely on hybridization of a target that is labelled with Cy3 fluorophore, or in the presence of an unlabelled target when a sandwich assay format is implemented with a labelled reporter oligonucleotide. Selectivity to determine the presence of mismatched sequences involves appropriate selection of nucleotide sequences to set melt temperatures, in conjunction with control of stringency conditions using formamide as a chaotrope. It was determined that both direct and sandwich assays on paper substrates are able to distinguish between wild-type and mutant-type samples.

Pharmacokinetic Profiling of Conjugated Therapeutic Oligonucleotides: A High-Throughput Method Based Upon Serial Blood Microsampling Coupled to Peptide Nucleic Acid Hybridization Assay.

Therapeutic oligonucleotides, such as small interfering RNAs (siRNAs), hold great promise for the treatment of incurable genetically defined disorders by targeting cognate toxic gene products for degradation. To achieve meaningful tissue distribution and efficacy in vivo, siRNAs must be conjugated or formulated. Clear understanding of the pharmacokinetic (PK)/pharmacodynamic behavior of these compounds is necessary to optimize and characterize the performance of therapeutic oligonucleotides in vivo. In this study, we describe a simple and reproducible methodology for the evaluation of in vivo blood/plasma PK profiles and tissue distribution of oligonucleotides. The method is based on serial blood microsampling from the saphenous vein, coupled to peptide nucleic acid hybridization assay for quantification of guide strands. Performed with minimal number of animals, this method allowed unequivocal detection and sensitive quantification without the need for amplification, or further modification of the oligonucleotides. Using this methodology, we compared plasma clearances and tissue distribution profiles of two different hydrophobically modified siRNAs (hsiRNAs). Notably, cholesterol-hsiRNA presented slow plasma clearances and mainly accumulated in the liver, whereas, phosphocholine-docosahexaenoic acid-hsiRNA was rapidly cleared from the plasma and preferably accumulated in the kidney. These data suggest that the PK/biodistribution profiles of modified hsiRNAs are determined by the chemical nature of the conjugate. Importantly, the method described in this study constitutes a simple platform to conduct pilot assessments of the basic clearance and tissue distribution profiles, which can be broadly applied for evaluation of new chemical variants of siRNAs and micro-RNAs.

A paper-based multiplexed resonance energy transfer nucleic acid hybridization assay using a single form of upconversion nanoparticle as donor and three quantum dots as acceptors

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reductive amination to evaluate the multiplexing capacity of luminescence resonance energy transfer (LRET) between UCNPs and quantum dots (QDs). This is the first account of a multiplexed bioassay strategy that demonstrates the principle of use of a single form of UCNP as donor and three different color emitting QDs as acceptors to concurrently determine three analytes. Broad absorbance profiles of green, orange and red QDs that spanned from the first exciton absorption peak to the UV region were in overlap with a blue emission band from UCNPs composed of NaYF4 that was doped with 30% Yb3+, 0.5% Tm3+, allowing for LRET that was stimulated using 980 nm near-infrared radiation. The characteristic narrow and well-defined emission peaks of UCNPs and QDs allowed for the collection of luminescence from each nanoparticle using a band-pass optical filter and an epi-fluorescence microscope. The LRET system was used for the concurrent detection of uidA, Stx1A and tetA gene fragments with selectivity even in serum samples, and reached limits of detection of 26 fmol, 56 fmol and 76 fmol, respectively.

Resonance Energy Transfer-Based Nucleic Acid Hybridization Assays on Paper-Based Platforms Using Emissive Nanoparticles as Donors.

Quantum dots (QDs) and upconverting nanoparticles (UCNPs) are luminescent nanoparticles (NPs) commonly used in bioassays and biosensors as resonance energy transfer (RET) donors. The narrow and tunable emissions of both QDs and UCNPs make them versatile RET donors that can be paired with a wide range of acceptors. Ratiometric signal processing that compares donor and acceptor emission in RET-based transduction offers improved precision, as it accounts for fluctuations in the absolute photoluminescence (PL) intensities of the donor and acceptor that can result from experimental and instrumental variations. Immobilizing NPs on a solid support avoids problems such as those that can arise with their aggregation in solution, and allows for facile layer-by-layer assembly of the interfacial chemistry. Paper is an attractive solid support for the development of point-of-care diagnostic assays given its ubiquity, low-cost, and intrinsic fluid transport by capillary action. Integration of nanomaterials with paper-based analytical devices (PADs) provides avenues to augment the analytical performance of PADs, given the unique optoelectronic properties of nanomaterials. Herein, we describe methodology for the development of PADs using QDs and UCNPs as RET donors for optical transduction of nucleic acid hybridization. Immobilization of green-emitting QDs (gQDs) on imidazole functionalized cellulose paper is described for use as RET donors with Cy3 molecular dye as acceptors for the detection of SMN1 gene fragment. We also describe the covalent immobilization of blue-emitting UCNPs on aldehyde modified cellulose paper for use as RET donors with orange-emitting QDs (oQDs) as acceptors for the detection of HPRT1 gene fragment. The data described herein is acquired using an epifluorescence microscope, and can also be collected using technology such as a typical electronic camera.

Creation of artificial luciferases to expand their analytical potential.

Bioluminescent proteins have been intensively used as high sensitive reporters in all kinds of binding assays (immuno-, nucleic acid hybridization assays, etc.) and in bioimaging. But natural luciferases do not always meet the requirements set for them as the assay reporters: thermostabitity, definite bioluminescence spectral and kinetics characteristics, stability to chemical modifications, etc. Luciferases with different appropriate characteristics as well as various luciferin derivatives were obtained using mutagenesis and chemical synthesis. Thanks to rigorous efforts of many researchers bioluminescencebased analytical techniques offer a great potential for solving analytical tasks in the field of biotechnology, biomedicine, pharmacology, etc.

Association of Nerine yellow stripe virus With Mosaic Disease of Crinum asiaticum Ornamental Plant in India

HomePlant DiseaseVol. 99, No. 11Association of Nerine yellow stripe virus With Mosaic Disease of Crinum asiaticum Ornamental Plant in India PreviousNext DISEASE NOTES OPENOpen Access licenseAssociation of Nerine yellow stripe virus With Mosaic Disease of Crinum asiaticum Ornamental Plant in IndiaS. Kumar, R. Raj, C. Kaur, and S. K. RajS. KumarSearch for more papers by this author, R. RajSearch for more papers by this author, C. KaurSearch for more papers by this author, and S. K. RajSearch for more papers by this authorAffiliationsAuthors and Affiliations S. Kumar R. Raj C. Kaur S. K. Raj , Plant Molecular Virology Laboratory, CPMB Division, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow-226001, Uttar Pradesh, India. Published Online:26 Aug 2015https://doi.org/10.1094/PDIS-02-15-0211-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Crinum asiaticum L. (Amaryllidaceae), commonly known as spider lily, is widely grown as an ornamental plant in gardens of tropical and subtropical countries for its long, strap-like, green leaves and clustered white flowers. During a survey in December 2013, severe mosaic symptoms were observed on a number of C. asiaticum plants growing in gardens at Noida and New Delhi cities of India. The symptoms were similar to those described earlier from Brazil on a potyvirus-infected Crinum sp. (Barboza et al. 2006); therefore, potyvirus infection was suspected. Electron microscopy was performed with leaf dip preparations of six randomly collected symptomatic leaf samples which revealed the presence of flexuous, rod-shaped, ∼700 × 12-nm virus particles, suggesting association of a potyvirus. These samples were further subjected to RT-PCR using potyvirus degenerate primers (Gibbs et al. 2003). RT-PCR resulted in the expected size (∼1.5 kb) amplicon in all symptomatic samples. The amplicons were cloned and sequenced, and sequence data were deposited as GenBank Accession Nos. KJ886933 (Nod-1), KM066970 (Nod-2), KM066968 (Nod-3), KJ886934 (Del-1), KM066971 (Del-2), and KM066969 (Del-3) isolates. Local alignments of the sequences of these isolates in NCBI revealed the presence of partial nuclear inclusion body (NIb), complete coat protein (CP) genes, and the 3′ UTR untranslated region (UTR) of a potyvirus. The isolates under study shared 98 to 99% sequence identity together and showed 78 to 99% identities with Nerine yellow stripe virus (NeYSV) isolates (FJ618537, DQ407932, EF362621, EF362622, EU042758, and JX865782) reported from New Zealand, The Netherlands, and USA. During phylogenetic analysis by MEGA (V.5.1) (Tamura et al. 2013), the isolates under study clustered together and showed close relationships with NeYSV isolates, and the potyvirus associated with mosaic disease of C. asiaticum was identified as NeYSV. The six and other nine symptomatic leaf samples of C. asiaticum were also tested by nucleic acid hybridization assay using the probe prepared from a clone of NeYSV (KJ886933), which gave strong signals of hybridization confirming presence of NeYSV in them. The probe may be used for the indexing of leaf samples collected from various locations and to understand the geographical distribution of NeYSV. A literature survey revealed the only report of association of an unidentified potyvirus with the mosaic disease of Crinum sp. from Brazil (Barboza et al. 2006) based on RT-PCR amplification of 2.0 kb product by potyvirus genus-specific primers. To best of our knowledge, this is the first report of natural occurrence of NeYSV on C. asiaticum in India. Because C. asiaticum is being propagated through bulbs, the dissemination of virus from nursery to nursery and from place to place is a major concern and now warrants the indexing of mother culture of C. asiaticum for NeYSV.

A Paper-Based Sandwich Format Hybridization Assay for Unlabeled Nucleic Acid Detection Using Upconversion Nanoparticles as Energy Donors in Luminescence Resonance Energy Transfer.

Bioassays based on cellulose paper substrates are gaining increasing popularity for the development of field portable and low-cost diagnostic applications. Herein, we report a paper-based nucleic acid hybridization assay using immobilized upconversion nanoparticles (UCNPs) as donors in luminescence resonance energy transfer (LRET). UCNPs with intense green emission served as donors with Cy3 dye as the acceptor. The avidin functionalized UCNPs were immobilized on cellulose paper and subsequently bioconjugated to biotinylated oligonucleotide probes. Introduction of unlabeled oligonucleotide targets resulted in a formation of probe-target duplexes. A subsequent hybridization of Cy3 labeled reporter with the remaining single stranded portion of target brought the Cy3 dye in close proximity to the UCNPs to trigger a LRET-sensitized emission from the acceptor dye. The hybridization assays provided a limit of detection (LOD) of 146.0 fmol and exhibited selectivity for one base pair mismatch discrimination. The assay was functional even in undiluted serum samples. This work embodies important progress in developing DNA hybridization assays on paper. Detection of unlabeled targets is achieved using UCNPs as LRET donors, with minimization of background signal from paper substrates owing to the implementation of low energy near-infrared (NIR) excitation.

A paper-based resonance energy transfer nucleic acid hybridization assay using upconversion nanoparticles as donors and quantum dots as acceptors

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reduction amination to develop a luminescence resonance energy transfer (LRET)-based nucleic acid hybridization assay. This first account of covalent immobilization of UCNPs on paper for a bioassay reports an optically responsive method that is sensitive, reproducible and robust. The immobilized UCNPs were decorated with oligonucleotide probes to capture HPRT1 housekeeping gene fragments, which in turn brought reporter conjugated quantum dots (QDs) in close proximity to the UCNPs for LRET. This sandwich assay could detect unlabeled oligonucleotide target, and had a limit of detection of 13fmol and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs, which are excellent LRET acceptors, demonstrated improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was attributed to the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The kinetics of hybridization were determined to be diffusion limited and full signal development occurred within 3min.

Polarization-Based DNA Sandwich Assay with Au Nanoparticles using the Influence of Inter-Particle Distance

Nucleic acid hybridization assay is widely used for application in biomedical research. In these field there is a considerable demand for the development of more sensitive, quantitative, rapid and low-cost method for target detection. To meet these requirements, Nanoparticle-based controlled assembly method via DNA has become a useful tool for clinical diagnostics because of their strict selectivity. Gold nanoparticles(AuNPs) can be synthesized amenably and can be made highly stable. Moreover, they have unique optical response of surface plasmon resonance and this property can be easily adjusted by varying their size, shape, and the surrounding chemical environment. In this research, we focus inter-particle distance of AuNP dimer. Inter-particle separations are greatly sensitive for the resonance wavelength and interaction between AuNP. When it is small(<2nm), it can be observed that specific optical response in occurred such as significantly high intensity scattering, peak shift of absorbance spectra.Recently, plasma mass spectrometry and surface enhanced raman spectroscopy(SERS) were reported as more sensitive method. However, they need sophisticated process and expensive instruments. Hence, We propose the method to use optical anisotropy and scattered light intensity as indices for accurate and robust discrimination between single AuNPs and dimers using single-particle polarization microscopy in aqueous solution. This method is called a direct light scattering(DLS) method. This is homogeneous, rapid low-cost, sensitive method. We tested several way to deduce distance between two AuNP in the step of fabricating AuNP dimers and evaluated these AuNPs to utilize DLS method in colloidal solution. In this presentation, we report the result and conclusion about the influence of inter-particle distance in our experiment.

Acceleration of Surface-Based Hybridization Reactions Using Isotachophoretic Focusing

We present a theoretical model and experimental demonstration of a novel method for acceleration of surface-based reactions using isotachophoresis (ITP). We use ITP to focus a sample of interest and deliver a high concentration target to a prefunctionalized surface, thus enabling rapid reaction at the sensor site. The concentration of the focused analyte is bound in space by the ITP interface and, upon reaction with the surface, continues electromigrating downstream, removing any contamination or reacted sample molecules from the surface. This constitutes a one-step react-and-wash assay which can be performed in a simple channel and does not require flow control elements or moving parts. We designed a novel microfluidic chip where reaction surfaces are formed by paramagnetic beads, immobilized at desired sites by an external magnetic field. Using this chip, we compared ITP-based surface hybridization to standard continuous flow-based hybridization and experimentally demonstrated a 2 orders of magnitude improvement in limit of detection (LoD) in a 3 min nucleic acid hybridization assay. The simple analytical model we present allows prediction of the rate of surface reaction under ITP and can be used to design and optimize such assays as a function of the physical properties of the system, including buffer chemistry, applied voltage, analyte mobility, analyte concentration, probe density, and surface length. The method, model, and experimental setup can be applied to various forms or surface reactions and may serve as the basis for highly genetic analysis and immunoassays.

Luminescence Resonance Energy Transfer-Based Nucleic Acid Hybridization Assay on Cellulose Paper with Upconverting Phosphor as Donors

A bioassay based on DNA hybridization on cellulose paper is a promising format for gene fragment detection that may be suited for in-field and rapid diagnostic applications. We demonstrate for the first time that luminescence resonance energy transfer (LRET) associated with upconverting phosphors (UCPs) can be used to develop a paper-based DNA hybridization assay with high sensitivity, selectivity and fast response. UCPs with strong green emission were synthesized and subsequently functionalized with streptavidin (UCP-strep). UCP-strep particles were immobilized on cellulose paper, and then biotinylated single-stranded oligonucleotide probes were conjugated onto the UCPs via streptavidin-biotin linkage. The UCPs served as donors that were LRET-paired with Cy3-labeled target DNA. Selective DNA hybridization enabled the proximity required for LRET-sensitized emission from Cy3, which was used as the detection signal. Hybridization was complete within 2 min, and the limit of detection of the method was 34 fmol, which is a significant improvement in comparison to an analogous fluorescence resonance energy transfer (FRET) assay based on quantum dots. The assay exhibited excellent resistance to nonspecific adsorption of noncomplementary short/long DNA and protein. The selectivity of the assay was further evaluated by one base pair mismatched (1BPM) DNA detection, where a maximum signal ratio of 3.1:1 was achieved between fully complementary and 1BPM samples. This work represents a preliminary but significant step for the development of paper-based UCP-LRET nucleic acid hybridization assays, which offer potential for lowering the limit of detection of luminescent hybridization assays due to the negligible background signal associated with optical excitation by near-infrared (NIR) light.

Application of enzyme bioluminescence for medical diagnostics.

Nowadays luciferases are effectively used as analytical instruments in a great variety of research fields. Of special interest are the studies dealing with elaboration of novel analytical systems for the purposes of medical diagnostics. The ever-expanding spectrum of clinically important analytes accounts for the increasing demand for new techniques for their detection. In this chapter we have made an attempt to summarize the results on applications of luciferases as reporters in binding assays including immunoassay, nucleic acid hybridization assay, and so on. The data over the last 15 years have been analyzed and clearly show that luciferase-based assays, due to extremely high sensitivity, low cost, and the lack of need for skilled personnel, hold much promise for clinical diagnostics.

The role of human papillomavirus infection in the head and neck region and methods for its detection.

Human papillomaviruses (HPV) are the etiological factors of many benign and malignant lesions localized in the skin and mucous membranes in the region of the urogenital organs and head and neck. Currently, most assays for the detection of HPV are based on detecting the presence of viral nucleic acids, mostly viral DNA. These molecular techniques can be divided into: 1) methods based on a targeted, selective amplification of nucleic acids, 2) signal amplification methods, 3) nucleic acid hybridization assays. This paper presents and explains a number of different HPV detection methods and provides examples of some commonly available commercial tests.