Anisotropy-based sensing with reference fluorophores

We describe a new approach to fluorescence sensing based on measurements of steady-state anisotropies in the presence of reference fluorophores with known anisotropies. The basic concept is that the anisotropy of a mixture reflects a weighted average of the anisotropies of the emitting species. By use of reference fluorophores the starting anisotropy can be near zero, or near 0.9 for oriented films which contain the reference fluorophore. Changing intensities of the analyte result in changes in anisotropy. A wide dynamic range of anisotropies is available because of the freedom to select high or low starting values. Anisotropy-based sensing was demonstrated for pH using 6-carboxyfluorescein and for protein affinity or immunoassay using an oriented film with high anisotropy and a protein labeled with a metal-ligand complex. The latter measurements were performed with a simple light-emitting diode excitation source without an excitation polarizer. The sensitive range of the assay can be adjusted by changing the intensity of the reference fluorophore. Anisotropy-based sensing can have numerous applications in clinical and analytical chemistry.     

Polarization sensing of fluorophores in tissues for drug compliance monitoring.

We used a new method, polarization sensing, to monitor the concentration of the fluorophore rhodamine 800 in an intralipid suspension and in chicken tissue. Rhodamine 800 (Rh800) could be excited at 648 nm using a laser pointer. We developed a simple device for measuring the combined emission from a highly polarized reference film and the unpolarized or orthogonally polarized emission of Rh800 from the scattering intralipid or tissue. The concentration of Rh800 in this medium was revealed by large changes in the polarization (P) with values of P ranging from 0.8 to -0.9. It is possible to vary the sensitive Rh800 concentration range by variation of the detected emission wavelengths, orientation of the excitation polarizer, or fluorophore concentration in the reference film. Polarization sensing of fluorophores in tissue requires only steady-state detection, and can be accomplished with simple and/or portable electronics. Such devices may find use in electronic detection of ingested medicines based on transdermal detection of nontoxic long-wavelength fluorophores.     

Modular tagging of amplicons using a single PCR for high‐throughput sequencing

High‐throughput sequencing (HTS) of PCR amplicons is becoming the method of choice to sequence one or several targeted loci for phylogenetic and DNA barcoding studies. Although the development of HTS has allowed rapid generation of massive amounts of DNA sequence data, preparing amplicons for HTS remains a rate‐limiting step. For example, HTS platforms require platform‐specific adapter sequences to be present at the 5′ and 3′ end of the DNA fragment to be sequenced. In addition, short multiplex identifier (MID) tags are typically added to allow multiple samples to be pooled in a single HTS run. Existing methods to incorporate HTS adapters and MID tags into PCR amplicons are either inefficient, requiring multiple enzymatic reactions and clean‐up steps, or costly when applied to multiple samples or loci (fusion primers). We describe a method to amplify a target locus and add HTS adapters and MID tags via a linker sequence using a single PCR. We demonstrate our approach by generating reference sequence data for two mitochondrial loci (COI and 16S) for a diverse suite of insect taxa. Our approach provides a flexible, cost‐effective and efficient method to prepare amplicons for HTS.     

Preparation of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes

Aptamers are single-stranded DNA or RNA molecules that can bind target molecules with high affinity and specificity. The conformation of an aptamer usually changes upon binding to its target analyte, and this property has been used in a wide variety of sensing applications, including detection based on fluorescence intensity, polarization, energy transfer, electrochemistry or color change. Colorimetric sensors are particularly important because they minimize or eliminate the necessity of using expensive and complicated instruments. Among the many colorimetric sensing strategies, metallic nanoparticle-based detection is desirable because of the high extinction coefficients and strong distance-dependent optical properties of the nanoparticles. Here, we describe a protocol for the preparation of aptamer-linked gold nanoparticle purple aggregates that undergo fast disassembly into red dispersed nanoparticles upon binding of target analytes. This method has proved to be generally applicable for colorimetric sensing of a broad range of analytes. The time range for the entire protocol is approximately 5 d, including synthesis and functionalization of nanoparticles, preparation of nanoparticle aggregates and sensing.     

Single Molecule Detection Technologies in Miniaturized High Throughput Screening: Fluorescence Correlation Spectroscopy

Fluorescence assay technologies used for miniaturized high throughput screening are broadly divided into two classes. Macroscopic fluorescence techniques (encompassing conventional fluorescence intensity, anisotropy [also often referred to as fluorescence polarization] and energy transfer) monitor the assay volume- and time-averaged fluorescence output from the ensemble of emitting fluorophores. In contrast, single-molecule detection (SMD) techniques and related approaches, such as fluorescence correlation spectroscopy (FCS), stochastically sample the fluorescence properties of individual constituent molecules and only then average many such detection events to define the properties of the assay system as a whole. Analysis of single molecular events is accomplished using confocal optics with an illumination/detection volume of approximately 1 fl (10(-15) L) such that the signal is insensitive to miniaturization of HTS assays to 1 μl or below. In this report we demonstrate the general applicability of one SMD technique (FCS) to assay configuration for target classes typically encountered in HTS and confirm the equivalence of the rate/equilibrium constants determined by FCS and by macroscopic techniques. Advantages and limitations of the current FCS technology, as applied here, and potential solutions, particularly involving alternative SMD detection techniques, are also discussed.     

A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays

The ability to identify active compounds (3hits2) from large chemical libraries accurately and rapidly has been the ultimate goal in developing high-throughput screening (HTS) assays. The ability to identify hits from a particular HTS assay depends largely on the suitability or quality of the assay used in the screening. The criteria or parameters for evaluating the 3suitability2 of an HTS assay for hit identification are not well defined and hence it still remains difficult to compare the quality of assays directly. In this report, a screening window coefficient, called 3Z-factor,2 is defined. This coefficient is reflective of both the assay signal dynamic range and the data variation associated with the signal measurements, and therefore is suitable for assay quality assessment. The Z-factor is a dimensionless, simple statistical characteristic for each HTS assay. The Z-factor provides a useful tool for comparison and evaluation of the quality of assays, and can be utilized in assay optimization and validation.     

Modulated fluorescence correlation spectroscopy with complete time range information

Two methods to combine fluorescence correlation spectroscopy (FCS) with modulated excitation, in a way that allows extraction of correlation data for all correlation times have been developed and experimentally verified. One method extracts distortion-free correlation data from measurements acquired with standard hardware correlators provided the fluorescence does not change systematically within the excitation pulses. This restriction does not apply to the second method, which, however, requires time-resolved acquisition of the fluorescence intensity. Modulation of the excitation in an FCS experiment is demonstrated to suppress triplet population buildup more efficiently than a corresponding reduction in continuous wave excitation intensity (shown for the dye rhodamine 6G in aqueous solution). Excitation modulation thus offers an additional means to optimize the FCS measurement conditions with respect to the photophysical properties of the dyes used. This possibility to suppress photoinduced states also provides a useful tool to distinguish additional processes occurring in the same time regime in the FCS measurements, as demonstrated here for the protonation kinetics of fluorescein at different pH. In general, the proposed concept opens for FCS measurements with a complete correlation timescale in a range of applications where a modulated excitation is either necessary or brings specific advantages.     

Microfluidic pH-sensing chips integrated with pneumatic fluid-control devices

This paper presents a microfluidic chip capable of performing precise continuous pH measurements in an automatic mode. The chip is fabricated using micro-electro-mechanical-systems (MEMS)-based techniques and incorporates polydimethylsiloxane (PDMS) microstructures, pH-sensing electrodes and pneumatic fluid-control devices. Through its enhanced microchannel design and use of pneumatic fluid-control devices, the microfluidic chip reduces the dead volume of the sample and increases the pumping rate. The maximum pumping rate of the developed micro-pump is 28 microL/min at an air pressure of 10 psi and a driving frequency of 10 Hz. The total sample volume consumed in each sensing operation is just 0.515 microL. As a result, the developed chip reduces the sample volume compared to conventional large-scale pH-sensing systems. The microfluidic chip employs the electrochemical sensing method to conduct precise pH level measurements. The sensing electrodes are fabricated by sputtering a layer of SiO(2)-LiO(2)-BaO-TiO(2)-La(2)O(3) (SLBTLO) onto platinum (Pt) electrodes and the pH value of the sample is evaluated by measuring the potential difference between the sensing electrodes and a reference electrode. Additionally, the integration of the microfluidic chip with a pneumatic fluid-control device facilitates automatic sample injection and a continuous sensing operation. The developed system provides a valuable tool with which to examine pH values in a wide range of biomedical and industrial applications.     

Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes

We studied fluorescence intensity, polarization and lifetime of some commonly used fluorophores conjugated to oligodeoxyribonucleotides with different primary and secondary structures. We found that fluorescence intensity can increase or decrease upon hybridization of the labeled strand to its complement depending on the sequence and position of the fluorophore. Up to 10-fold quenching of the fluorescence upon hybridization was observed when the dye moiety was attached close to the 3′ end and the 3′-terminal base was either dG or dC. No quenching upon hybridization was observed when the dye was positioned within the same sequence context but close to the 5′ end. The presence of a dG overhang quenches the fluorescence less efficiently than a blunt end dG-dC or dC-dG base pair. When located internally in the double strand, the dG-dC base pair does not affect the fluorescence of the nearby dye. Guanosine in a single-stranded oligonucleotide quenches the fluorescence of nearby dye by <2-fold. Upon duplex formation, this quenching is eliminated and the fluorescence increases. This increase can only be detected when the fluorophore is located at least 6 nt from the terminal dG-dC base pair. The change of fluorescence polarization upon duplex formation inversely correlates with the change of intensity. Fluorescein conjugated to a single-stranded oligonucleotide or a duplex undergoes a bi-exponential decay with ~4 and ~1 ns lifetimes.     

Non-Faradaic electrochemical detection of protein interactions by integrated neuromorphic CMOS sensors

Electronic detection of the binding event between biotinylated bovine serum albumen (BSA) and streptavidin is demonstrated with the chemoreceptive neuron MOS (CnuMOS) device. Differing from the ion-sensitive field-effect transistors (ISFET), CnuMOS, with the potential of the extended floating gate determined by both the sensing and control gates in a neuromorphic style, can provide protein detection without requiring analyte reference electrodes. In comparison with the microelectrode arrays, measurements are gathered through purely capacitive, non-Faradaic interactions across insulating interfaces. By using a (3-glycidoxypropyl)trimethoxysilane (3-GPS) self-assembled monolayer (SAM) as a simple covalent link for attaching proteins to a silicon dioxide sensing surface, a fully integrated, electrochemical detection platform is realized for protein interactions through monotone large-signal measurements or small-signal impedance spectroscopy. Calibration curves were created to coordinate the sensor response with ellipsometric measurements taken on witness samples. By monitoring the film thickness of streptavidin capture, a sensitivity of 25ng/cm2 or 2A of film thickness was demonstrated. With an improved noise floor the sensor can detect down to 2ng/(cm2mV) based on the calibration curve. AC measurements are shown to significantly reduce long-term sensor drift. Finally, a noise analysis of electrochemical data indicates 1/f(alpha) behavior with a noise floor beginning at approximately 1Hz.     

Comparison of mid-infrared Fourier transform vibrational circular dichroism measurements with single and dual polarization modulations

Fourier transform infrared vibrational circular dichroism (FTIR-VCD) measurements have gone through major advances in the last decade. A major thrust in these advances was to find ways that can minimize the VCD spectral artifacts and obtain the VCD signals at enhanced signal quality. However, all these advances are not incorporated in FTIR-VCD instruments manufactured by some commercial manufacturers. In this article, we compare the measurements obtained with single and dual polarization modulations to seek the attention of the users of single polarization modulation method in minimizing the artifacts in such measurements. We also report that the VCD spectrum of a home-made collagen film can serve as a simple and convenient sample for routine verification of the correct functioning of the VCD spectrometers.     

Prevention,diagnosis and treatment of high‐throughput sequencing data pathologies

High‐throughput sequencing (HTS) technologies generate millions of sequence reads from DNA/RNA molecules rapidly and cost‐effectively, enabling single investigator laboratories to address a variety of ‘omics’ questions in nonmodel organisms, fundamentally changing the way genomic approaches are used to advance biological research. One major challenge posed by HTS is the complexity and difficulty of data quality control (QC). While QC issues associated with sample isolation, library preparation and sequencing are well known and protocols for their handling are widely available, the QC of the actual sequence reads generated by HTS is often overlooked. HTS‐generated sequence reads can contain various errors, biases and artefacts whose identification and amelioration can greatly impact subsequent data analysis. However, a systematic survey on QC procedures for HTS data is still lacking. In this review, we begin by presenting standard ‘health check‐up’ QC procedures recommended for HTS data sets and establishing what ‘healthy’ HTS data look like. We next proceed by classifying errors, biases and artefacts present in HTS data into three major types of ‘pathologies’, discussing their causes and symptoms and illustrating with examples their diagnosis and impact on downstream analyses. We conclude this review by offering examples of successful ‘treatment’ protocols and recommendations on standard practices and treatment options. Notwithstanding the speed with which HTS technologies – and consequently their pathologies – change, we argue that careful QC of HTS data is an important – yet often neglected – aspect of their application in molecular ecology, and lay the groundwork for developing a HTS data QC ‘best practices’ guide.     

Fluorescence properties of rhodamine 800 in whole blood and plasma

We have characterized the fluorescence spectral properties of rhodamine 800 (Rh800) in plasma and blood in order to test the possibility of making clinical fluorescence measurements in whole blood without separation steps. Rh800 was used because of its absorption at red/near-infrared wavelengths away from the absorption bands of hemoglobin. We utilized the front-face illumination and detection to minimize the effects of absorption and/or scatter during measurements. The presence of Rh800 was detected in plasma and blood using steady-state fluorescence measurements. Absorption due to hemoglobin reduced the Rh800 intensity from the blood. Fluorescence lifetime measurements in plasma and blood showed that it is possible to recover lifetime parameters of Rh800 in these media. We obtained mean lifetimes of 1.90 and 1.86 ns for Rh800 in plasma and blood, respectively. Using the recently described modulation sensing method, we quantified the concentrations of Rh800 in plasma and blood. Rh800 was detected at a concentration of as low as 2 microM in both media. High anisotropy values were obtained for Rh800 in plasma and blood using steady-state and anisotropy decay measurements, implying the tight binding of this probe to the contents of these media. This binding can be exploited to monitor the concentrations of different blood components using already existing or new red-emitting probes that will be specially designed to bind to these components with high specificity. To test this possibility of direct measurements in blood, we used Rh800 to monitor albumin in the presence of red blood cells. Increase in the polarization of Rh800 as the concentration of albumin was increased in the presence of the red cells showed the feasibility of such measurements.     

Two-dimensional fluorescence intensity distribution analysis: theory and applications

A method of sample analysis is presented which is based on fitting a joint distribution of photon count numbers. In experiments, fluorescence from a microscopic volume containing a fluctuating number of molecules is monitored by two detectors, using a confocal microscope. The two detectors may have different polarizational or spectral responses. Concentrations of fluorescent species together with two specific brightness values per species are determined. The two-dimensional fluorescence intensity distribution analysis (2D-FIDA), if used with a polarization cube, is a tool that is able to distinguish fluorescent species with different specific polarization ratios. As an example of polarization studies by 2D-FIDA, binding of 5'-(6-carboxytetramethylrhodamine) (TAMRA)-labeled theophylline to an anti-theophylline antibody has been studied. Alternatively, if two-color equipment is used, 2D-FIDA can determine concentrations and specific brightness values of fluorescent species corresponding to individual labels alone and their complex. As an example of two-color 2D-FIDA, binding of TAMRA-labeled somatostatin-14 to the human type-2 high-affinity somatostatin receptors present in stained vesicles has been studied. The presented method is unusually accurate among fluorescence fluctuation methods. It is well suited for monitoring a variety of molecular interactions, including receptors and ligands or antibodies and antigens.     

Theory of light quenching: effects of fluorescence polarization, intensity, and anisotropy decays.

Experimental studies have recently demonstrated that fluorescence emission can be quenched by laser light pulses from modern high repetition rate lasers, a phenomenon we call "light quenching." We now describe the theory of light quenching and some of its effects on the steady-state and time-resolved intensity and anisotropy decays of fluorophores. Light quenching can decrease or increase the steady-state or time-zero anisotropy. Remarkably, the light quenching can break the usual z axis symmetry of the excited-state population, and the emission polarization can range from -1 to +1 under selected conditions. The measured anisotropy (or polarization) depends upon whether the observation axis is parallel or perpendicular to the propagation direction of the light quenching beam. The effects of light quenching are different for a single pulse, which results in both excitation and quenching, as compared with a time-delayed quenching pulse. Time-delayed light quenching pulses can result in step-like changes in the time-dependent intensity or anisotropy and are predicted to cause oscillations in the frequency-domain intensity and anisotropy decays. The increasing availability of pulsed laser sources offers the opportunity for a new class of two-pulse or multiple-pulse experiments where the sample is prepared by an excitation pulse, the excited state population is modified by the quenching pulse(s), followed by time- or frequency-domain measurements of the resulting emission.     

Microfluidic devices for sample preparation and rapid detection of foodborne pathogens

Rapid detection of foodborne pathogens at an early stage is imperative for preventing the outbreak of foodborne diseases, known as serious threats to human health. Conventional bacterial culturing methods for foodborne pathogen detection are time consuming, laborious, and with poor pathogen diagnosis competences. This has prompted researchers to call the current status of detection approaches into question and leverage new technologies for superior pathogen sensing outcomes. Novel strategies mainly rely on incorporating all the steps from sample preparation to detection in miniaturized devices for online monitoring of pathogens with high accuracy and sensitivity in a time-saving and cost effective manner. Lab on chip is a blooming area in diagnosis, which exploits different mechanical and biological techniques to detect very low concentrations of pathogens in food samples. This is achieved through streamlining the sample handling and concentrating procedures, which will subsequently reduce human errors and enhance the accuracy of the sensing methods. Integration of sample preparation techniques into these devices can effectively minimize the impact of complex food matrix on pathogen diagnosis and improve the limit of detections. Integration of pathogen capturing bio-receptors on microfluidic devices is a crucial step, which can facilitate recognition abilities in harsh chemical and physical conditions, offering a great commercial benefit to the food-manufacturing sector. This article reviews recent advances in current state-of-the-art of sample preparation and concentration from food matrices with focus on bacterial capturing methods and sensing technologies, along with their advantages and limitations when integrated into microfluidic devices for online rapid detection of pathogens in foods and food production line.     

High Throughput Screening with Multiphoton Excitation

Fluorescence detection is extensively used in high throughput screening. In HTS there is a continuous migration toward higher density plates and smaller sample volumes. In the present report we describe the advantages of two-photon or multiphoton excitation for HTS. Multiphoton excitation (MPE) is the simultaneous absorption of two long-wavelength photons to excite the lowest singlet state of the fluorophore. MPE is typically accomplished with short but high-intensity laser pulses, which allows simultaneous absorption of two or more photons. The intensity of the multiphoton-induced fluorescence is proportional to the square, cube, or higher power of the instantneous photon flux. Consequently, two-photon or multiphoton excitation only occurs at the focal point of the incident beam. This property of two-photon excitation allows the excited volume to be very small and to be localized in the center of each well in the HTS plate. We show that two-photon-induced fluorescence of fluorescein can be reliably measured in microwell plates. We also show the use of 6-carboxy fluorescein as a pH probe with two-photon excitation, and measure 4'-6-diamidino-2-phenylindole (DAPI) binding and two-photon-induced fluorescence. In further studies we measure the time-dependent intensity decays of DAPI bound to DNA and of calcium-dependent fluorophores. Finally, we demonstrate the possibility of three-photon excitation of several fluorophores, including indole, in the HTS plate. These results suggest that MPE can be used in high-density multiwell plates.     

jackalope: A swift,versatile phylogenomic and high‐throughput sequencing simulator

High‐throughput sequencing (HTS) is central to the study of population genomics and has an increasingly important role in constructing phylogenies. Choices in research design for sequencing projects can include a wide range of factors, such as sequencing platform, depth of coverage and bioinformatic tools. Simulating HTS data better informs these decisions, as users can validate software by comparing output to the known simulation parameters. However, current standalone HTS simulators cannot generate variant haplotypes under even somewhat complex evolutionary scenarios, such as recombination or demographic change. This greatly reduces their usefulness for fields such as population genomics and phylogenomics. Here I present the R package jackalope that simply and efficiently simulates (i) sets of variant haplotypes from a reference genome and (ii) reads from both Illumina and Pacific Biosciences platforms. Haplotypes can be simulated using phylogenies, gene trees, coalescent‐simulation output, population‐genomic summary statistics, and Variant Call Format (VCF) files. jackalope can simulate single, paired‐end or mate‐pair Illumina reads, as well as reads from Pacific Biosciences. These simulations include sequencing errors, mapping qualities, multiplexing and optical/PCR duplicates. It can read reference genomes from fasta files and can simulate new ones, and all outputs can be written to standard file formats. jackalope is available for Mac, Windows and Linux systems.