Aptamer binding assays for proteins: The thrombin example—A review

Experimentally selected single-stranded DNA and RNA aptamers are able to bind to specific target molecules with high affinity and specificity. Many analytical methods make use of affinity binding between the specific targets and their aptamers. In the development of these methods, thrombin is the most frequently used target molecule to demonstrate the proof-of-principle. This paper critically reviews more than one hundred assays that are based on aptamer binding to thrombin. This review focuses on homogeneous binding assays, electrochemical aptasensors, and affinity separation techniques. The emphasis of this review is placed on understanding the principles and unique features of the assays. The principles of most assays for thrombin are applicable to the determination of other molecular targets.     

Self-referencing a single waveguide grating sensor in a micron-sized deep flow chamber for label-free biomolecular binding assays

In order to allow the design of increasingly sensitive label-free biosensors, compensation of environmental fluctuations is emerging as the dominant hurdle. The system and technique presented here utilize a unique combination of microfluidics, optical instrumentation, and image processing to provide a reference signal for each label-free biomolecular binding assay. Moreover, this reference signal is generated from the same sensor used to detect the biomolecular binding events. In this manner, the reference signal and the binding signal share nearly all common-mode noise sources (temperature, pressure, vibration, etc.) and their subtraction leaves the purest binding signal possible. Computational fluid dynamic simulations have been used to validate the flow behavior and thermal characteristics of the fluids inside the sensing region. This system has been demonstrated in simple bulk refractive index tests, as well as small molecule (biotin/streptavidin) binding experiments. The ability to perform not only simple binding but also control experiments has been discussed, indicating the wide applicability of the technique.     

Photochemically amplified detection of molecular recognition events: an ultra-sensitive fluorescence turn-off binding assay

Amplified fluorescence quenching methodology based on massive autocatalytic photo-unmasking of a dual function sensitizer-quencher is developed and adopted for photoassisted ultra-sensitive detection of molecular recognition events. The resulting binding assay, based on a molecular recognition-triggered photo-amplified cascade with concomitant decrease of fluorescence is validated with the biotin-avidin pair, achieving attomolar detection.     

Fluorescein labeling of estrogen for application of fluorescence polarization binding assays for antibody and receptor

The fluorescence polarization binding assay (FPBA) using fluorescein-labeled estrogen tracer is a homogeneous assay applicable to both estrogen antibody and estrogen receptor-binding assays. Two estrogen-ethylendiamine fluoresceinthiobamyl (E-EDF) tracers were synthesized; estrogen-6-EDF (E-6-F) derived from 6-ketoestradiol 6-(o-carboxymethyl) oxime and estrogen-17-EDF (E-17-F) was from 17β-estradiol 17-hemisuccinate. In both FPBAs using antibody and receptor, E-6-F tracer (Rf_365nm=0.58) showed a better binding response than E-17-F (Rf_365nm=0.70) indicating that the 17-position of estrogen seems to play an essential role as a binding site for antibody or receptor. In the optimized conditions of FPBA for E₂ using E-6-F tracer, antibody binding (K_d=9.4×10⁻⁹ M) is 50 times sensitive than receptor binding (K_d=4.6×10⁻⁸ M). Binding responses of estrogen and its related chemicals by FPBA indicate that antibody binding assay is able to screen the structural similarity of estrogen showing some response with methyltestosterone (K_i=2.1×10⁻⁵ M). On the other hand, the receptor assay is able to screen for estrogenic chemicals such as tamoxifen (K_i=4.5×10⁻⁹ M) and diethylstilbesterol (K_i=8.1×10⁻⁷ M). Therefore, E-6-F tracer is useful as a tracer for FPBA that is able to screen for chemicals structurally similar to estrogen using antibody, and that is able to screen for chemicals functionally similar to estrogen using receptor binding assay.     

Radioimmunoassays and 2-site immunoradiometric "sandwich" assays: basic principles

The "sandwich" or noncompetitive reagent-excess, 2-site immunoradiometric assay (2-site IRMA), ELISA, USERIA, and related techniques, have several advantages compared with the traditional or competitive radioimmunoassays. IRMAs can provide improved sensitivity and specificity. However, IRMAs present some practical problems with nonspecific binding, increased consumption of antibody, biphasic dose response curve, (high dose hook effect), and may require special techniques for dose response curve analysis. We anticipate considerable growth in the popularity and importance of 2-site IRMA.     

Determining protease substrate selectivity and inhibition by label-free supramolecular tandem enzyme assays

An analytical method has been developed for the continuous monitoring of protease activity on unlabeled peptides in real time by fluorescence spectroscopy. The assay is enabled by a reporter pair comprising the macrocycle cucurbit[7]uril (CB7) and the fluorescent dye acridine orange (AO). CB7 functions by selectively recognizing N-terminal phenylalanine residues as they are produced during the enzymatic cleavage of enkephalin-type peptides by the metalloendopeptidase thermolysin. The substrate peptides (e.g., Thr-Gly-Ala-Phe-Met-NH(2)) bind to CB7 with moderately high affinity (K ≈ 10(4) M(-1)), while their cleavage products (e.g., Phe-Met-NH(2)) bind very tightly (K > 10(6) M(-1)). AO signals the reaction upon its selective displacement from the macrocycle by the high affinity product of proteolysis. The resulting supramolecular tandem enzyme assay effectively measures the kinetics of thermolysin, including the accurate determination of sequence specificity (Ser and Gly instead of Ala), stereospecificity (d-Ala instead of l-Ala), endo- versus exopeptidase activity (indicated by differences in absolute fluorescence response), and sensitivity to terminal charges (-CONH(2) vs -COOH). The capability of the tandem assay to measure protease inhibition constants was demonstrated on phosphoramidon as a known inhibitor to afford an inhibition constant of (17.8 ± 0.4) nM. This robust and label-free approach to the study of protease activity and inhibition should be transferable to other endo- and exopeptidases that afford products with N-terminal aromatic amino acids.     

Nanoporous niobium oxide for label-free detection of DNA hybridization events

We found that DNA probes can be immobilized on anodically prepared porous niobium oxide without a chemical modification of both the DNA probes and the substrate. By using the porous niobium oxide with a positive surface charge, DNA hybridization events are detected on the basis of the blue-shift of a maximum absorption peak in UV-vis-NIR spectroscopy. The blue-shift is ascribed to the change of surface charge upon single- or double-stranded DNA. The method does not require a label and shows high sensitivity with the detection limit of the concentration of 1nM.     

Aptamer biosensor for label-free impedance spectroscopy detection of proteins based on recognition-induced switching of the surface charge

The recognition of proteins by aptamer-modified electrode transducers reverses the surface charge and leads to a novel label-free impedance spectroscopy bioelectronic detection protocol based on a decrease in the electron transfer resistance.     

Micromechanical measurement of AChBP binding for label-free drug discovery

A potential binding assay based on binding-driven micromechanical motion is described. Acetylcholine binding protein (AChBP) was used to modify a microcantilever. The modified microcantilever was found to bend on application of the naturally occurring agonist (acetylcholine) or the antagonist (nicotine and d-tubocurarine). Control experiments show that microcantilevers modified without AChBP do not respond to acetylcholine, nicotine, and d-tubocurarine. K(d) values obtained for acetylcholine, nicotine, and d-tubocurarine are similar to those obtained from radio-ligand binding assays. These results suggest that the microcantilever system has potential for use in label free, drug screening applications.     

AC electrothermal enhancement of heterogeneous assays in microfluidics

AC-driven electrothermal flow is used to enhance the temporal performance of heterogeneous immuno-sensors in microfluidic systems by nearly an order of magnitude. AC electrokinetic forces are used to generate electrothermal flow, which in turn produces a circular stirring fluid motion that enhances the transport of diffusion-limited proteins. This provides more binding opportunities between suspended antigens and wall-immobilized antibodies. We investigate experimentally the effectiveness of electrothermal stirring, using a biotin-streptavidin heterogeneous assay, in which biotin is immobilized, and fluorescently-labeled streptavidin is suspended in a high conductivity buffer (sigma = 1.0 S m(-1)). Microfabricated electrodes were integrated within a microwell and driven at a frequency of f= 200 kHz and 10 V(rms). Fluorescent intensity measurements show that for a five minute assay, electrothermal stirring increases the binding rate by a factor of almost nine. Similar binding improvement was measured for longer assays, up to fifteen minutes. The electrothermal enhancement of this assay was modeled numerically and agrees with experimental binding rates. The measured fluid velocity of 22 +/- 2 microm s(-1) was significantly lower than that predicted by the numerical model, 1.1 mm s(-1), but nevertheless shows the same fourth power dependence on applied potential. The results demonstrate the ability for electrothermal stirring to reliably improve the response time and sensitivity within a given time limit for microfluidic diffusion-limited sensors.     

Monitoring on-chip Pictet-Spengler reactions by integrated analytical separation and label-free time-resolved fluorescence

High-throughput screening for optimal reaction conditions and the search for efficient catalysts is of eminent importance in the development of chemical processes and for expanding the spectrum of synthetic methodologies in chemistry. In this context we report a novel approach for a microfluidic chemical laboratory integrating organic synthesis, separation and time-resolved fluorescence detection on a single microchip. The feasibility of our integrated laboratory is demonstrated by monitoring the formation of tetrahydroisoquinoline derivatives by Pictet-Spengler condensation. After on-chip reaction the products and residual starting material were separated enantioselectively on the same chip. On-chip deep UV laser-induced fluorescence detection with time-correlated single photon counting was applied for compound assignment. The system was utilized to screen reaction conditions and various substrates for Pictet-Spengler reactions on-chip. Finally, the microlab was successfully applied to investigate enantioselective reactions using BINOL-based phosphoric acids as organocatalysts.     

In-silico investigations in heterogeneous catalysis--combustion and synthesis of small alkanes

In this critical review, we cover first-principles density functional calculations relevant to alkane oxidation and synthesis over transition metal catalysts. For oxidation, we focus upon Pt, Rh, Pd and Ni surfaces, while for synthesis we consider Co, Ru, Fe and Ni. Throughout, we emphasise the insight to be gained by thinking of each kind of reaction as the inverse of the other, with the directionality determined simply by the choice of metal catalyst and the reaction conditions. We highlight particularly the role of low-coordination sites (steps, kinks, etc.) and the emerging consensus over the importance of the formyl intermediate in facilitating the rate-determining step (249 references).     

A label-free G-quadruplex-based switch-on fluorescence assay for the selective detection of ATP

A G-quadruplex-based, label-free, switch-on fluorescence detection method has been developed for the selective detection of ATP in aqueous solution using crystal violet as a G-quadruplex-selective probe. The assay is highly simple and rapid, and does not require the use of fluorescent labeling.     

Supramolecular displacement-mediated activation of a silent fluorescence probe for label-free ligand screening

We report a new approach for the rapid screening of analyte binding affinities for a target protein. We demonstrate that a molecular probe, with a pro-fluorophore substrate and ligand moieties, can be hindered from enzymatic access when bound to the target protein. When analytes displace the probe from the protein's binding pocket, a fluorescence profile is generated. This profile is used to discriminate analytes based on their relative binding affinities.     

Silver nanoparticle-enhanced fluorescence in microtransponder-based immuno- and DNAhybridization assays

The aim of this study is to improve assay sensitivity in common solid-phase bioassay configurations as the result of using silver nanoparticles. The solid phase was provided by numerically indexed, silicon-based electronic chips, microtransponders (p-Chips) that have previously been used in multiplexed assays. Assay configurations investigated included an ELISA-type immunoassay and a DNA hybridization assay. The surface of p-Chips was derivatized with the silver island film (SIF) and a polymer, and then characterized with AFM and SEM. Silver nanoparticle sizes were in the range of 100 to 200 nm. Four fluorophores were tested for fluorescence enhancement; namely, green fluorescent protein, phycoerythrin, Cy3 and Alexa Fluor 555. We consistently observed significant fluorescence enhancement and sensitivity improvement in the p-Chip-based assays: the sensitivity in the cytokine IL-6 immunoassay was 4.3 pg/ml, which represented a 25-fold increase over the method not involving a SIF; and 50 pM in the hybridization assay, a 38-fold increase. The greatest enhancement was obtained for p-Chip surfaces derivatized first with the polymer and then coated with SIF. In conclusion, we show that the SIF-p-Chip-based platform is a highly sensitive method to quantify low-abundance biomolecules in nucleic acid-based assays and immunoassays.     

NMR investigations into heterogeneous structures of thermosensitive microgel particles

The internal properties of submicron poly(N‐isopropylmethacrylamide) latex particles were investigated as a function of the methylene bisacrylamide (MBA) concentration used as a crosslinker. Two experimental approaches were performed. First, quasi‐electric light scattering measurements provided the size variation of the particles as a function of temperature, from which the swelling capacity of the particles as a function of MBA were estimated. In addition, the broadening and lowering effects of the volume phase transition temperature were detected from the turbidity of the solutions versus the MBA concentration. Second, observations of the transverse relaxation of protons gave evidence for heterogeneous structures inside the particles; several structural parts were discriminated from one another from different proton mobilities detected through magnetic relaxation rates. Corresponding to the concentration gradients of the crosslinker, the internal particle structures were looser and looser from the core to the shell. The state of the gelation of the polymer particles was governed by the initial amount of the crosslinker introduced into the latex recipe. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 889–898, 2000     

Development and application of fluorescence polarization assays in drug discovery

Fluorescence polarization technology has been used in basic research and commercial diagnostic assays for many decades, but has begun to be widely used in drug discovery only in the past six years. Originally, FP assays for drug discovery were developed for single-tube analytical instruments, but the technology was rapidly converted to high-throughput screening assays when commercial plate readers with equivalent sensitivity became available. This review will discuss fluorescence polarization assays in current use in drug discovery research as well as those in development that will likely be used in the near future. These assays include targets such as kinases, phosphatases, proteases, G-protein coupled receptors, and nuclear receptors.     

Optimization of DNA-tagged dye-encapsulating liposomes for lateral-flow assays based on sandwich hybridization

A novel protocol for the synthesis of dye-encapsulating liposomes tagged with DNA oligonucleotides at their outer surface was developed. These liposomes were optimized for use as signal enhancement agents in lateral-flow sandwich-hybridization assays for the detection of single-stranded RNA and DNA sequences. Liposomes were synthesized using the reverse-phase evaporation method and tagged with oligonucleotides by adding cholesteryl-modified DNA probes to the initial lipid mixture. This resulted in a greatly simplified protocol that provided excellent control of the probe coverage on the liposomes and cut the preparation time from 16 hours to just 6 hours. Liposomes were prepared using probe concentrations ranging from 0.00077 to 0.152 mol% of the total lipid, several hydrophobic and polyethylene glycol-based spacers between the cholesteryl anchor and the probe, and liposome diameters ranging from 208 nm to 365 nm. The liposomes were characterized by dynamic light scattering, visible spectroscopy, and fluorescence spectroscopy. Their signal enhancement functionality was compared by using them in lateral-flow optical biosensors for the detection of single-stranded DNA sequences. In these assays, an optimal reporter probe concentration of 0.013 mol%, liposome diameter of 315 nm, and liposome optical density of 0.4–0.6 at 532 nm were found. The spacer length between the cholesteryl anchor and the probe showed no significant effect on the signals in the lateral-flow assays. The results presented here provide important data for the general use of liposomes as labels in analytical assays, with specific emphasis on nucleic acid detection via lateral flow assays.