Oil-sealed femtoliter fiber-optic arrays for single molecule analysis

This paper describes a novel method for fabricating and sealing high-density arrays of femtoliter reaction chambers. We chemically etch one end of a 2.3 mm diameter glass optical fiber bundle to create an array of microwells. We then use a contact printing method to selectively modify the surface of the material between microwells with a hydrophobic silane. This modification makes it possible to fill the wells with aqueous solution and then seal them with a droplet of oil, forming an array of isolated reaction chambers. Individual β-galactosidase molecules trapped in these reaction chambers convert a substrate into a fluorescent product that can be readily detected because a high local concentration of product is achieved. This binary readout can be used for ultra-sensitive measurements of enzyme concentration. We observed that the percentage of wells showing enzyme activity was linearly dependent on the concentration of soluble β-galactosidase in the picomolar range. A similar response was also observed for streptavidin-β-galactosidase captured by biotinylated beads. These arrays are also suitable for performing single-molecule kinetics studies on hundreds to thousands of enzyme molecules simultaneously. We observed a broad distribution of catalytic rates for individual β-galactosidase molecules trapped in the microwells, in agreement with previous studies using similar arrays that were mechanically sealed. We have further demonstrated that this femtoliter fiber-optic array can be integrated into a PDMS microfluidic channel system and sealed with oil on-chip, creating an easy to use and high-throughput device for single-molecule analysis.     

Detection of single-molecule DNA hybridization using enzymatic amplification in an array of femtoliter-sized reaction vessels

Improving the sensitivity of DNA biosensors is extremely important in clinical diagnostics, gene therapy, and a variety of other biomedical studies. In this regard, we have developed a highly sensitive single molecule DNA assay platform with a 1fM experimental detection limit using enzymatic amplification in an array of femtoliter-sized reaction wells. To validate the utility of this technology in our study, we employed a fiber optic array to create thousands of femtoliter-sized reaction wells, each specifically functionalized with oligonucleotide probes capable of capturing biotinylated target DNA. After hybridization, the fiber was incubated with streptavidin-labeled enzyme solution. The bound single enzyme molecules were confined to individual reaction vessels containing excess fluorogenic substrate and catalyzed the production of a sufficient number of fluorescent product molecules to generate a detectable signal. At low target DNA concentrations with relatively short incubation times, only a small percentage of the capture sites bind target DNA, enabling a binary readout of target concentration from the high-density fiber array. This simple binary readout-based scheme is easy to perform and exhibits a high signal-to-noise ratio in the presence of trace amounts of DNA target. Furthermore, it also should be possible to extend this technology to protein detection by modifying the reaction wells with specific capture antibodies. We expect this assay to be useful in a number of biomedical applications where accurate and highly sensitive target analysis is critical.     

SERS as tool for the analysis of DNA-chips in a microfluidic platform

A sequence-specific detection method of DNA is presented combining a solid chip surface for immobilisation of capture DNAs with a microfluidic platform and a readout of the chip based on SERS. The solid chip surface is used for immobilisation of different capture DNAs, where target strands can be hybridised and unbound surfactants can be washed away. For the detection via SERS, short-labelled oligonucleotides are hybridised to the target strands. This technique is combined with a microfluidic platform that enables a fast and automated preparation process. By applying a chip format, the problems of sequence-specific DNA detection in solution phase by means of SERS can be overcome. With this setup, we are able to distinguish between different complementary and non-complementary target sequences in one sample solution.     

A Simple and Highly Sensitive Electrochemical Biosensor for microRNA Detection Using Target‐Assisted Isothermal Exponential Amplification Reaction

A simple and highly sensitive electrochemical biosensor for microRNA (miRNA) detection was successfully developed by integrating a target‐assisted isothermal exponential amplification reaction (EXPAR) with enzyme‐amplified electrochemical readout. The binding of target miRNA with the immobilized linear DNA template generated a part duplex and triggered primer extension reaction to form a double‐stranded DNA. Then one of the DNA strands was cleaved by nicking endonuclease and extended again. The short fragments with the same sequence as the target miRNA except for the replacement of uridines and ribonucleotides with thymines and deoxyribonucleotides could be displaced and released. Hybridization of these released DNA fragments with other amplification templates and their extension on the templates led to target exponential amplification. Integrating with enzyme‐amplified electrochemical readout, the electrochemical signal decreases with the increasing target microRNA concentration. The method could detect miRNA down to 98.9 fM with a linear range from 100 fM to 10 nM. The fabrication and binding processes were characterized with cyclic voltammetry and electrochemical impedance spectroscopy. The specificity of the method allowed single‐nucleotide difference between miRNA family members to be discriminated. The established biosensor displayed excellent analytical performance toward miRNA detection and might present a powerful and convenient tool for biomedical research and clinic diagnostic application.     

Polymeric integrated selective enrichment target (ISET) for solid-phase-based sample preparation in MALDI-TOF MS

A polymer microfabricated proteomic sample preparation and MALDI MS sample presentation device, the integrated selective enrichment target (ISET), comprising an array of perforated nanovials is reported. Each perforated nanovial can be filled with selective extraction media (microbeads) for purification and concentration of protein/peptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The main areas covered are the influence of the molding-process-induced surface roughness and how to address the lack of inherent conductivity in the polyetheretherketone (PEEK) material for optimal MALDI MS readout. Application of the disposable polymeric ISET devices for solid-phase extraction and phosphopeptide capture is also demonstrated.     

基于G-四链体-血红素和链式放大反应的基因检测传感器的构建

该文以特殊设计的DNA序列为捕获探针,以G-四链体-血红素复合物作为信号分子,利用链式反应实现目标DNA的灵敏检测。在目标DNA存在时,捕获探针与目标DNA相互识别,同时目标DNA能与辅助探针发生连续的链式反应,从而在电极表面引入大量G-四链体结构。血红素存在下,G-四链体可与血红素结合形成具有很强电化学信号的G-四链体-血红素复合物。用差分脉冲伏安法(DPV)扫描得到的电化学信号与体系中的目标DNA浓度存在对应关系,从而实现对目标DNA的检测。在各组分浓度最适的情况下,电流响应值与目标DNA浓度在0.01～10 pmol/L内具有良好的线性关系,检出限可达8 fmol/L。该传感器灵敏度高、特异性好,具有良好的应用前景。     

Point-of-care detection of Microcystin-LR with a personal glucose meter in drinking water source

In this study, we described a point-of-care sensing protocol for rapid and sensitive detection of Microcystin-LR (MC-LR) in water by personal glucose meter. The POCT method possessed good reproducibility, selectivity, and stability, which may have potential for many other on-site detection applications.     

Quantitation of femtomolar protein levels via direct readout with the electrochemical proximity assay

We have developed a separation-free, electrochemical assay format with direct readout that is amenable to highly sensitive and selective quantitation of a wide variety of target proteins. Our first generation of the electrochemical proximity assay (ECPA) is composed of two thrombin aptamers which form a cooperative complex only in the presence of target molecules, moving a methylene blue (MB)-conjugated oligonucleotide close to a gold electrode. Without washing steps, electrical current is increased in proportion to the concentration of a specific target protein. By employing a DNA-based experimental model with the aptamer system, we show that addition of a short DNA competitor can reduce background current of the MB peak to baseline levels. As such, the detection limit of aptamer-based ECPA for human thrombin was 50 pM via direct readout. The dual-probe nature of ECPA gave high selectivity and 93% recovery of signal from 2.5 nM thrombin in 2% bovine serum albumin (BSA). To greatly improve the flexibility of ECPA, we then proved the system functional with antibody-oligonucleotide conjugates as probes; the insulin detection limit was 128 fM with a dynamic range of over 4 orders of magnitude in concentration, again with high assay selectivity. ECPA thus allows separation-free, highly sensitive, and highly selective protein detection with a direct electrochemical readout. This method is extremely flexible, capable of detecting a wide variety of protein targets, and is amenable to point-of-care protein measurement, since any target with two aptamers or antibodies could be assayed via direct electrochemical readout.     

Label-Free Impedance Biosensors: Opportunities and Challenges

Impedance biosensors are a class of electrical biosensors that show promise for point-of-care and other applications due to low cost, ease of miniaturization, and label-free operation. Unlabeled DNA and protein targets can be detected by monitoring changes in surface impedance when a target molecule binds to an immobilized probe. The affinity capture step leads to challenges shared by all label-free affinity biosensors; these challenges are discussed along with others unique to impedance readout. Various possible mechanisms for impedance change upon target binding are discussed. We critically summarize accomplishments of past label-free impedance biosensors and identify areas for future research.     

A single-molecule enzymatic assay in a directly accessible femtoliter droplet array

The enzyme assay in a femtoliter chamber array is a simple and efficient method for concentrating the reaction product; it greatly improves the detection sensitivity down to the single-molecule level. However, in previous methods, controlling the initiation and termination of the reaction in each chamber is difficult once enclosed. Furthermore, the recovery of the enzyme and product is also difficult. To overcome these drawbacks, we developed a femtoliter droplet array in which the individual droplets are fixed on the substrate and are directly accessible from outside. A hydrophilic-in-hydrophobic micropatterned surface was used for the preparation of the droplets. When the aqueous solution on the surface is exchanged with oil, the hydrophilic surface retains the aqueous solution, and more than 10(6) dome-shaped droplets that are usable for further assay can be prepared simultaneously. The curvature radius of the droplet obeys the Young-Laplace equation, and the volume can be precisely controlled by the micropipette, which applies pressure into the droplet. Changing the pressure makes the addition, collection, and exchange of the aqueous content for individual droplets possible. Using these advantages, we successfully measured the kinetic parameters of the single-molecule enzyme β-galactosidase and rotary motor protein F(1)-ATPase enclosed in a droplet.     

Label-free electrical detection of pyrophosphate generated from DNA polymerase reactions on field-effect devices

We introduce a label-free approach for sensing polymerase reactions on deoxyribonucleic acid (DNA) using a chelator-modified silicon-on-insulator field-effect transistor (SOI-FET) that exhibits selective and reversible electrical response to pyrophosphate anions. The chemical modification of the sensor surface was designed to include rolling-circle amplification (RCA) DNA colonies for locally enhanced pyrophosphate (PPi) signal generation and sensors with immobilized chelators for capture and surface-sensitive detection of diffusible reaction by-products. While detecting arrays of enzymatic base incorporation reactions is typically accomplished using optical fluorescence or chemiluminescence techniques, our results suggest that it is possible to develop scalable and portable PPi-specific sensors and platforms for broad biomedical applications such as DNA sequencing and microbe detection using surface-sensitive electrical readout techniques.     

Hybridization of DNA to bead-immobilized probes confined within a microfluidic channel

We report the factors influencing the capture of DNA by DNA-modified microbeads confined within a microfluidic channel. Quantitative correlation of target capture efficiency to probe surface concentration, solution flow rate, and target concentration are discussed. The results indicate that the microfluidic system exhibits a limit of detection of approximately 10(-10) M (approximately 10(-16) mol) DNA and a selectivity factor of approximately 8 x 10(3). Typical hybridization times are on the order of minutes.     

A new spectrophotometric readout for the alanine-triphenyl tetrazolium chloride system for high-dose dosimetry

The electron spin resonance (ESR) readout of radical concentration in irradiated L-alanine is well known as a transfer dosimetry system, however, the high cost of the ESR equipment is a serious handicap for large-scale routine application. In this study the reaction between irradiated L-alanine powder and aqueous solution of triphenyl tetrazolium chloride (TTC) was studied. The radical produced by irradiation of alanine reacts during the dissolution with TTC forming pink-colored formazans with absorption maxima around 401 and 487 nm. This readout method can be used in the 2–200 kGy dose range.     

Affinity Selections of DNA-Encoded Chemical Libraries on Carbonic Anhydrase IX-Expressing Tumor Cells Reveal a Dependence on Ligand Valence

DNA-encoded chemical libraries are typically screened against purified protein targets. Recently, cell-based selections with encoded chemical libraries have been described, commonly revealing suboptimal performance due to insufficient recovery of binding molecules. We used carbonic anhydrase IX (CAIX)-expressing tumor cells as a model system to optimize selection procedures with code-specific quantitative polymerase chain reaction (qPCR) as selection readout. Salt concentration and performing PCR on cell suspension had the biggest impact on selection performance, leading to 15-fold enrichment factors for high-affinity monovalent CAIX binders (acetazolamide; K_D=8.7 nM). Surprisingly, the homobivalent display of acetazolamide at the extremities of both complementary DNA strands led to a substantial improvement of both ligand recovery and enrichment factors (above 100-fold). The optimized procedures were used for selections with a DNA-encoded chemical library comprising 1 million members against tumor cell lines expressing CAIX, leading to a preferential recovery of known and new ligands against this validated tumor-associated target. This work may facilitate future affinity selections on cells against target proteins which might be difficult to express otherwise.     

Electrochemical biosensors for detection of point mutation based on surface ligation reaction and oligonucleotides modified gold nanoparticles

An electrochemical method for point mutation detection based on surface ligation reaction and oligonucleotides (ODNs) modified gold nanoparticles (AuNPs) was demonstrated. Point mutation identification was achieved using Escherichia coli DNA ligase. This system for point mutation detection relied on a sandwich assay comprising capture ODN immobilized on Au electrodes, target ODN and ligation ODN. Because of the sequence-specific surface reactions of E. coli DNA ligase, the ligation ODN covalently linked to the capture ODN only in the presence of a perfectly complementary target ODN. The presence of ligation products on Au electrode was detected using chronocoulometry through hybridization with reporter ODN modified AuNPs. The use of AuNPs improved the sensitivity of chronocoulometry in this approach, a detection limit of 0.9 pM complementary ODN was obtained. For single base mismatched ODN (smODN), a negligible signal was observed. Even if the concentration ratio of complementary ODN to smODN was decreased to 1:1000, a detectable signal was observed. This work may provide a specific, sensitive and cost-efficient approach for point mutant detection.     

Electron capture atmospheric pressure photoionization mass spectrometry: analysis of fullerenes, perfluorinated compounds, and pentafluorobenzyl derivatives

An electron capture (EC) ionization mechanism has been found to be highly efficient in negative-ion atmospheric pressure photoionization (APPI) for the analysis of compounds with positive electron affinity (EA). Using negative-ion APPI, we first report the sensitive detection of natural electrophores with limited polarity, such as fullerenes and perfluorinated compounds, by mass spectrometry (MS). Using direct infusion on a quadrupole time-of-flight (QTOF) mass spectrometer, the limits of detection (LODs) for C(60) and perfluoromethylcyclohexane were determined to be 0.15 pg (0.2 fmol) and 1 femtoliter (fL) ( approximately 1.5 pg or 4.3 fmol), respectively. As the EA of the analyte increases, the detection sensitivity is enhanced. Making use of the accurate mass measurement capability of the QTOF mass spectrometer, we were able to investigate the elemental composition of the ions in each spectrum and attribute the observed high sensitivity to an EC-initiated ionization process. The proposed EC ionization mechanism is further supported by the observation of a dissociative EC reaction of pentafluorobenzyl (PFB)-derivatized phenols. The analysis of phenols by EC-APPI of their PFB derivatives resulted in very high sensitivity, with the lowest reported LOD of approximately 0.17 pg (0.5 fmol) being for 2,4-dinitrophenol. For future LC/EC-APPI-MS applications, the effect of additives and solvents on sensitivity was also tested and reported.     

Label-free DNA hybridization detection and single base-mismatch discrimination using CE-ICP-MS assay

Detecting a specific DNA sequence and discriminating single base-mismatch is critical to clinical diagnosis, paternity testing, forensic sciences, food and drug industry, pathology, genetics, environmental monitoring, and anti-bioterrorism. To this end, capillary electrophoresis (CE) coupled with the inductively coupled plasma mass spectrometry (ICP-MS) method is developed using the displacing interaction between the target ssDNA and the competitor Hg(2+) for the first time. The thymine-rich capture ssDNA 1 is interacted with the competitor Hg(2+), forming an assembled complex in a hairpin-structure between the thymine bases arrangement at both sides of the capture ssDNA 1. In the presence of a target ssDNA with stronger affinity than that of the competitor Hg(2+), the energetically favorable hybridization between capture ssDNA 1 and the target ssDNA destroys the hairpin-structure and releases the competitor as free Hg(2+), which was then read out and accurately quantified by CE-ICP-MS assay. Under the optimal CE separation conditions, free Hg(2+) ions and its capture ssDNA 1 adduct were baseline separated and detected on-line by ICP-MS; the increased peak intensity of free Hg(2+) against the concentration of perfectly complementary target ssDNA was linear over the concentration range of 30-600 nmol L(-1) with a limit of detection of 8 nmol L(-1) (3s, n = 11) in the pre-incubated mixture containing 1 μmol L(-1) Hg(2+) and 0.2 μmol L(-1) capture ssDNA 1. This new assay method is simple in design since any target ssDNA binding can in principle result in free Hg(2+) release by 6-fold Hg(2+) signal amplification, avoiding oligonucleotide labeling or assistance by excess signal transducer and signal reporter to read out the target. Due to element-specific detection of ICP-MS in our assay procedure, the interference from the autofluorescence of substrata was eliminated.     

MALDI-TOF MS as a label-free approach to rapid inhibitor screening

Mass Spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays. These measurements are typically performed by time-consuming LC-MS to eliminate buffer salts that interfere with electrospray ionization MS. However, matrix-assisted laser desorption ionization, time-of-flight MS (MALDI-TOF MS) offers a label-free and direct readout of substrate and product, a fast sampling rate, and is tolerant of many buffer salts, reagents, and compounds that are typically found in enzyme reaction mixtures. In this report, a demonstration of how MALDI-TOF MS can be used to directly measure ratios of substrates and products to produce IC(50) curves for rapid enzyme assays and compound screening is provided. Typical reproducibility parameters were <7% RSD-a value comparable to ESI MS quantitative assays and well within the acceptable limits for screening assays. The speed of the MALDI readout is currently about 10 s per sample, thus allowing for over 7500 samples/day. From a simplicity standpoint, the enzymatic reaction mixtures are prepared by liquid handling robots, the reactions are stopped by addition of a 10 times volume of acidic matrix solution, and the samples are simultaneously transferred to MALDI target plate for analysis. Importantly, the ratios of substrate to product are of sufficient reproducibility to eliminate the need for internal standards and, thus, minimize the cost and increasing the speed of assay development.     

Array-based capture, distribution, counting and multiplexed assaying of beads on a centrifugal microfluidic platform

We present a novel centrifugal microfluidic platform for the highly efficient manipulation and analysis of particles for applications in bead-based assays. The platform uses an array of geometrical V-cup barriers to trap particles using stopped-flow sedimentation under highly reproducible hydrodynamic conditions. The impact parameters governing the occupancy distribution and capture efficiency of the arrayed traps are investigated. The unique, nearly 100% capture efficiency paired with the capability to establish sharply peaked, single occupancy distributions enables a novel, digital readout mode for color-multiplexed, particle-based assays with low-complexity instrumentation. The presented technology marks an essential step towards a versatile platform for the integration of bead- and cell-based biological assays.     

An integrated microfluidic processor for single nucleotide polymorphism-based DNA computing

An integrated microfluidic processor is developed that performs molecular computations using single nucleotide polymorphisms (SNPs) as binary bits. A complete population of fluorescein-labeled DNA "answers" is synthesized containing three distinct polymorphic bases; the identity of each base (A or T) is used to encode the value of a binary bit (TRUE or FALSE). Computation and readout occur by hybridization to complementary capture DNA oligonucleotides bound to magnetic beads in the microfluidic device. Beads are loaded into sixteen capture chambers in the processor and suspended in place by an external magnetic field. Integrated microfluidic valves and pumps circulate the input DNA population through the bead suspensions. In this example, a program consisting of a series of capture/rinse/release steps is executed and the DNA molecules remaining at the end of the computation provide the solution to a three-variable, four-clause Boolean satisfiability problem. The improved capture kinetics, transfer efficiency, and single-base specificity enabled by microfluidics make our processor well-suited for performing larger-scale DNA computations.