Beating the reaction limits of biosensor sensitivity with dynamic tracking of single binding events

The clinical need for ultrasensitive molecular analysis has motivated the development of several endpoint-assay technologies capable of single-molecule readout. These endpoint assays are now primarily limited by the affinity and specificity of the molecular-recognition agents for the analyte of interest. In contrast, a kinetic assay with single-molecule readout could distinguish between low-abundance, high-affinity (specific analyte) and high-abundance, low-affinity (nonspecific background) binding by measuring the duration of individual binding events at equilibrium. Here, we describe such a kinetic assay, in which individual binding events are detected and monitored during sample incubation. This method uses plasmonic gold nanorods and interferometric reflectance imaging to detect thousands of individual binding events across a multiplex solid-phase sensor with a large area approaching that of leading bead-based endpoint-assay technologies. A dynamic tracking procedure is used to measure the duration of each event. From this, the total rates of binding and debinding as well as the distribution of binding-event durations are determined. We observe a limit of detection of 19 fM for a proof-of-concept synthetic DNA analyte in a 12-plex assay format.First author draf

A digital microarray using interferometric detection of plasmonic nanorod labels

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and dynamic range of traditional fluorescence microarrays compared to other techniques have been the technology's Achilles' Heel, and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule ('digital') regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform's primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about three orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10x objective lens. This approach does not require any chemical enhancement such as silver deposition, and scans arrays with a throughput similar to commercial fluorescence devices. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about one million directly from a single scan

A digital microarray using interferometric detection of plasmonic nanorod labels

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and dynamic range of traditional fluorescence microarrays compared to other techniques have been the technology's Achilles' Heel, and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule ('digital') regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform's primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about three orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10x objective lens. This approach does not require any chemical enhancement such as silver deposition, and scans arrays with a throughput similar to commercial fluorescence devices. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about one million directly from a single scan.First author draf

Robust visualization and discrimination of nanoparticles by interferometric imaging

Single-molecule and single-nanoparticle biosensors are a growing frontier in diagnostics. Digital biosensors are those which enumerate all specifically immobilized biomolecules or biological nanoparticles, and thereby achieve limits of detection usually beyond the reach of ensemble measurements. Here we review modern optical techniques for single nanoparticle detection and describe the single-particle interferometric reflectance imaging sensor (SP-IRIS). We present challenges associated with reliably detecting faint nanoparticles with SP-IRIS, and describe image acquisition processes and software modifications to address them. Specifically, we describe a image acquisition processing method for the discrimination and accurate counting of nanoparticles that greatly reduces both the number of false positives and false negatives. These engineering improvements are critical steps in the translation of SP-IRIS towards applications in medical diagnostics.R01 AI096159 - NIAID NIH HHSFirst author draf

Digital microarrays: single-molecule readout with interferometric detection of plasmonic nanorod labels

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and limited dynamic range of traditional fluorescence microarrays compared to other detection techniques have been the technology’s Achilles’ heel and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule (“digital”) regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform’s primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about 3 orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10× objective lens. This approach does not require any chemical signal enhancement such as silver deposition and scans arrays with a throughput similar to commercial fluorescence scanners. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about 6 orders of magnitude directly from a single scan. As a proof-of-concept digital protein microarray assay, we demonstrated detection of hepatitis B virus surface antigen in buffer with a limit of detection of 3.2 pg/mL. More broadly, the technique’s simplicity and high-throughput nature make digital microarrays a flexible platform technology with a wide range of potential applications in biomedical research and clinical diagnostics.The authors wish to thank Oguzhan Avci and Jacob Trueb for thoughtful comments and suggestions regarding numerical optimization of the optical system. This work was funded in part by a research contract with ASELSAN, Inc. and the Wallace H. Coulter Foundation 2010 Coulter Translational Award. (ASELSAN, Inc.; Wallace H. Coulter Foundation Coulter Translational Award)Accepted manuscrip

Bankers at the gate : microfinance and the high cost of borrowed logics

In this paper we examine how the interaction between influences of commercial banking and poverty alleviation shaped the evolution of modern microfinance. Using institutional theory as a lens, we observe that the commercial banking logic increasingly displaced the microfinance field's foundational poverty alleviation and development principles over time. We argue that this process of displacement can occur inadvertently as organizations that embody multiple logics draw disproportionately on only one of those logics when developing legitimating accounts of their activity to stakeholders. Furthermore, we introduce the concept of permeability – the extent to which the elements of a logic are ambiguous and loosely coupled – to explain why some logics may be more or less open to the influence of other logics. We conclude by discussing implications for entrepreneurship and poverty alleviation more generally

Tradition in organizations : a custodianship framework

The study of tradition has become increasingly important in management research explaining phenomena as diverse as socialization, identity, institutional maintenance, and field-level change. Whereas recent studies bring new insights, management scholars’ conceptualization of tradition suffers from a lack of theoretical integration. In this article, we identify the major perspectives on tradition used in the literature and propose an integrative “custodianship framework” that encourages researchers to examine stability and change in organizational traditions by considering the perspectives, interests, and power of custodians surrounding a tradition over time. We suggest that future research explicitly consider the importance of place as both the rootedness and emplacement of traditions motivate the need for custodianship

Multiregional Satellite Precipitation Products Evaluation over Complex Terrain

An extensive evaluation of nine global-scale high-resolution satellite-based rainfall (SBR) products is performed using a minimum of 6 years (within the period of 2000-13) of reference rainfall data derived from rain gauge networks in nine mountainous regions across the globe. The SBR products are compared to a recently released global reanalysis dataset from the European Centre for Medium-Range Weather Forecasts (ECMWF). The study areas include the eastern Italian Alps, the Swiss Alps, the western Black Sea of Turkey, the French Cévennes, the Peruvian Andes, the Colombian Andes, the Himalayas over Nepal, the Blue Nile in East Africa, Taiwan, and the U.S. Rocky Mountains. Evaluation is performed at annual, monthly, and daily time scales and 0.25° spatial resolution. The SBR datasets are based on the following retrieval algorithms: Tropical Rainfall Measuring Mission Multisatellite Precipitation Analysis (TMPA), the NOAA/Climate Prediction Center morphing technique (CMORPH), Precipitation Estimation from Remotely Sensed Information Using Artificial Neural Networks (PERSIANN), and Global Satellite Mapping of Precipitation (GSMaP). SBR products are categorized into those that include gauge adjustment versus unadjusted. Results show that performance of SBR is highly dependent on the rainfall variability. Many SBR products usually underestimate wet season and overestimate dry season precipitation. The performance of gauge adjustment to the SBR products varies by region and depends greatly on the representativeness of the rain gauge network

Experimental assessment of substrate storage yield

Çevre biyoteknolojisindeki son gelişmeler doğrultusunda aktif çamur sistemlerinde substrat depolama kavramı önemli bir proses olarak kabul edilmektedir. Substrat depolama kavramı, aktif çamur modellemesine, Aktif Çamur Modeli No.3 (ASM3) ve saf substratlar için önerilen biyokimyasal modellerle dâhil edilmiştir. Depolama kavramının ortaya konması, modele çok fazla serbestlik derecesi sağlayarak, modeli daha karmaşık bir hale getiren pek çok kinetik ve stokiyometrik katsayının da eklenmesine yol açmıştır. Substrat depolama dönüşüm oranı, YSTO, ASM3 modelindeki en önemli parametrelerden biridir. Depolama dönüşüm oranı, substratın stokiyometrik olarak ne kadarının depolama ürünlerine dönüşeceğini belirleyen etkin bir model bileşenidir. Bu çalışma, ASM3 modelinde tanımlanan substrat depolama dönüşüm oranının (YSTO), deneysel olarak belirlenmesini amaçlamaktadır. Önerilen yöntem, atıksularda depolama ürünlerinin miktarlarının belirlenmesini gerektirmeyen, respirometrik bir prosedürdür. Yöntemde, model simulasyonlarından yararlanılarak, depolama prosesi için tüketilen oksijen miktarının oksijen tüketim hızı (OTH) eğrisi üzerinde belirlenmesinin esasları ortaya konmuştur. Önerilen yaklaşım kesikli deneylerle elde edilen oksijen tüketim hızına (OTH) dayanmaktadır. Yöntem substrat depolaması için kullanılan oksijen miktarının hesaplanması için grafiksel bir metot tanımlamaktadır. Yöntemin değerlendirilmesi için farklı deneysel başlangıç koşullarının kullanıldığı model simülasyonlarından yararlanılmıştır. Önerilen yöntem ile YSTO değeri %2’den daha düşük hata oranları ile belirlenebilmiştir. Deneysel yöntem ile, asetat, glikoz ve evsel atıksuyun kullanıldığı ve farklı F/M oranlarında yürütülen respirometrik testler sonucunda, depolama dönüşüm oranları, asetat için 0.78 gKOİ/gKOİ, glikoz için 0.87 gKOİ/gKOİ ve evsel atıksu için 0.96 gKOİ/gKOİ olarak belirlenmiştir. Anahtar Kelimeler: Asetat, Aktif Çamur Modeli No.3, evsel atıksu, glikoz, respirometri, depolama.Substrate storage under dynamic conditions is recently regarded as a significant process for activated sludge systems. The dynamic conditions and substrate gradients convey activated sludge cultures to develop a storage response when external substrate is present in the system. Substrate storage is incorporated into activated sludge modeling with Activated Sludge Model No. 3 (ASM3) and with biochemical models for pure substrates. ASM3 has been proposed for activated sludge systems both for aerobic and anoxic conditions. Introducing storage phenomena has also introduced a number of stoichiometric and kinetic coefficients making the model rather complicated with many degrees of freedom. Some default values have been proposed, but calibration of kinetic and stoichiometric parameters is needed for various applications. The storage yield (YSTO) is one of the most important parameters of the model, since it represents the stoichiometric amount of substrate converted into storage products, which are subsequently utilized for growth. The assessment of YSTO is therefore crucial for the accurate estimation of the overall electron acceptor utilization and sludge production. The information on the magnitude of the storage yield was mostly derived from pure culture studies. ASM3 suggested value of YSTO as 0.85 mgCOD/mgCOD under aerobic  conditions for domestic sewage. An experimental procedure was developed for the respirometric determination of bacterial storage yield (YSTO) as defined in the Activated Sludge Model No. 3. The proposed approach is based on the oxygen utilization rate (OUR) profile obtained from a batch test and correlates the area under the OUR curve to the amount of oxygen associated with substrate storage. The procedure is based on respirometry and does not involve measurement of storage products, as it will not always be possible or reliable to determine the amount of all the storage products when a complex substrate such as domestic sewage is concerned. It is theoretically possible to compute substrate storage yield (YSTO) if the oxygen used for the storage of a known amount of readily biodegradable COD can be determined by means of respirometric measurements. Such measurements however only provide the total oxygen utilization rate (OUR) of the system and not the OUR specific for the process of interest alone. Thus, it is necessary to understand and interpret the components of a total OUR versus time curve associated with the utilization of a readily biodegradable substrate. Model simulation was used to evaluate the procedure for different initial experimental conditions. Obtained results indicated that the proposed procedure was quite consistent with model-input values, involving an error of less than 2%, aside from analytical errors associated with standard COD measurements, for tests to be conducted with feeding (F/M) ratios over 0.1 gCOD/g cellCOD. The procedure was used to determine the storage yield, YSTO, associated with acetate, glucose and domestic sewage, together with mixtures of acetate/glucose and acetate/domestic sewage at different initial F/M ratios. YSTO was calculated as 0.78 gCOD/gCOD for acetate, 0.87 gCOD/gCOD for glucose and 0.96 gCOD/gCOD for domestic sewage. The high YSTO level related to domestic sewage, consistently obtained for a wide range of initial F/M ratios, challenges the validity of the concept of SS in ASM3, which is defined as the biodegradable fraction of the soluble substrate and tested in the study. The assumption introduced with ASM3 that the entire soluble biodegradable COD could be regarded as readily biodegradable substrate, although valid for pure substrates, requires, as in this case, careful evaluation for wastewaters with more complex substrate compositions. If readily biodegradable substrate is actually less, the corresponding oxygen consumption can only be interpreted with a superficially higher YSTO. The experiments conducted on substrate mixtures confirmed the validity of YSTO values calculated for individual substrates, yielding a transient pattern reflecting the character of the dominant substrate fraction in the mixture. For glucose/acetate mixtures, they provided a clear indication of a faster storage rate for glucose as compared to acetate. The proposed procedure may also be used to calculate initial readily biodegradable COD concentration, SS1, for a generally adopted YSTO value, as it defines a stoichiometric procedure between the storage yield and the available readily biodegradable substrate. Keywords: Acetate, Activated Sludge Model No.3, domestic sewage, glucose, respirometry, storage

Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing

Background: High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. Results: We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. Conclusions: The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons