Reusable electrochemical sensing platform for highly sensitive detection of small molecules based on structure-switching signaling aptamers

Aptamers are nucleic acids that have high affinity and selectivity for their target molecules. A target may induce the structure switching from a DNA/DNA duplex to a DNA/target complex. In the present study, a reusable electrochemical sensing platform based on structure-switching signaling aptamers for highly sensitive detection of small molecules is developed using adenosine as a model analyte. A gold electrode is first modified with polytyramine and gold nanoparticles. Then, thiolated capture probe is assembled onto the modified electrode surface via sulfur-gold affinity. Ferrocene (Fc)-labeled aptamer probe, which is designed to hybridize with capture DNA sequence and specifically recognize adenosine, is immobilized on the electrode surface by hybridization reaction. The introduction of adenosine triggers structure switching of the aptamer. As a result, Fc-labeled aptamer probe is forced to dissociate from the sensing interface, resulting in a decrease in redox current. The decrement of peak current is proportional to the amount of adenosine. The present sensing system could provide both a wide linear dynamic range and a low detection limit. In addition, high selectivity, good reproducibility, stability, and reusability are achieved. The recovery test demonstrates the feasibility of the designed sensing system for an adenosine assay.     

Aptameric peptide for one-step detection of protein kinase

Protein kinases are significant regulators in the cell signal pathway, and it is difficult to achieve quick kinase detection because traditional kinase assays normally rely on a time-consuming kinase phosphorylation process. Herein, we present a novel one-step strategy to detect protein kinase by using a kinase-specific aptameric peptide-functionalized quartz crystal microbalance (QCM) electrode, in which the detection can be finished in less than 10 min. A peptide kinase inhibitor (IP(20)) was used as the aptameric peptide because of its selective and strong interaction with the target protein kinase (cyclic adenosine monophosphate-dependent protein kinase A, PKA), high stability, and ease of inexpensive synthesis, presenting a new direct recognition element for kinase. The aptameric peptide was immobilized on the Au-coated quartz electrode through dual-thiol anchoring and the binding of His-tagged peptide with a nitrilotriacetic acid/Ni(II) complex, fabricating a highly specific and stable detection platform. The interaction of aptameric peptide with kinase was monitored with the QCM in real time, and the concentration of protein kinase was sensitively measured by the frequency response of the QCM with the low detection limit for PKA at 0.061 mU μL(-1) and a linear range from 0.64 to 22.33 mU μL(-1). This method is rapid and reagentless and does not require a phosphorylation process. The versatility of our aptameric peptide-based strategy has also been demonstrated by the application in kinase assay using electrochemical impedance spectroscopy. Moreover, this method was successfully applied to detect the forskolin/3-isobutyl-1-methylxanthine-stimulated activation of PKA in cell lysate.     

Nanotube-based colorimetric probe for ultrasensitive detection of ataxia telangiectasia mutated protein

We have developed a nanotube-based colorimetric probe using multiwalled carbon nanotubes (MWNTs), anti-immunoglobulin G (anti-IgG), and horseradish peroxidase (HRP). The probe was used as an alternative to conventional colorimetric conjugates to obtain amplified signals in a sandwich-type immunoassay for ataxia telangiectasia mutated (ATM), a potential biomarker for radiation doses and cancers. Results show that the MWNT-based probe colorimetry was 5000 times more sensitive than a conventional ELISA, while its concentration range was 10,000 times wider than that of the latter. Its limit of detection (LOD) was 0.2 fg/mL (54 aM, ~32 molecules in 1 μL samples). Control experiments showed that detection of ATM molecules at the picogram-level could still be achieved in samples that contained protein makers present at more than 100 times the ATM concentration, demonstrating the high specificity of the technique. The MWNT-based probe also has the potential to become a universal probe for colorimetric assays of most protein markers because it can recognize the associated rabbit polyclonal antibodies.     

Mass amplifying probe for sensitive fluorescence anisotropy detection of small molecules in complex biological samples

Fluorescence anisotropy (FA) is a reliable and excellent choice for fluorescence sensing. One of the key factors influencing the FA value for any molecule is the molar mass of the molecule being measured. As a result, the FA method with functional nucleic acid aptamers has been limited to macromolecules such as proteins and is generally not applicable for the analysis of small molecules because their molecular masses are relatively too small to produce observable FA value changes. We report here a molecular mass amplifying strategy to construct anisotropy aptamer probes for small molecules. The probe is designed in such a way that only when a target molecule binds to the probe does it activate its binding ability to an anisotropy amplifier (a high molecular mass molecule such as protein), thus significantly increasing the molecular mass and FA value of the probe/target complex. Specifically, a mass amplifying probe (MAP) consists of a targeting aptamer domain against a target molecule and molecular mass amplifying aptamer domain for the amplifier protein. The probe is initially rendered inactive by a small blocking strand partially complementary to both target aptamer and amplifier protein aptamer so that the mass amplifying aptamer domain would not bind to the amplifier protein unless the probe has been activated by the target. In this way, we prepared two probes that constitute a target (ATP and cocaine respectively) aptamer, a thrombin (as the mass amplifier) aptamer, and a fluorophore. Both probes worked well against their corresponding small molecule targets, and the detection limits for ATP and cocaine were 0.5 μM and 0.8 μM, respectively. More importantly, because FA is less affected by environmental interferences, ATP in cell media and cocaine in urine were directly detected without any tedious sample pretreatment. Our results established that our molecular mass amplifying strategy can be used to design aptamer probes for rapid, sensitive, and selective detection of small molecules by means of FA in complex biological samples.     

Unmodified gold nanoparticles as a colorimetric probe for visual methamphetamine detection

In recent years, in addition to the classic drugs, addiction to a series of new drug classes known as club drugs has increased significantly. Fast and low-cost bioassay for the detection of amphetamine-based drugs can be an effective strategy towards reducing their abuse. In this study, we designed a sensitive bioassay strategy using gold nanoparticles (GNPs) and the aptamers that possess high affinity toward methamphetamine (MA). It is suggested that the aptamer adopts different tertiary structures in the presence and/or absence of its specific target and GNPs can effectively differentiate between these two states by their characteristic surface plasmon resonance-based colour change. Visual detection of MA and 3,4-methylenedioxy-N-methylamphetamine (MDMA) in the low micromolar range is possible within minutes with the use of this method.     

Comparison of glycosphingolipids and antibodies as receptor molecules for ricin detection

Glycosphingolipids (GSLs) have been shown to undergo strong interactions with a number of protein toxins, including potential bioterrorism agents such as ricin and botulinum neurotoxin. Characterization of this interaction in recent years has led to a number of studies where GSLs were used as the recognition molecules for biosensing applications. Here, we offer a comparison of quartz crystal microbalance (QCM) sensors for the detection of ricin using antibodies and the GSLs GM1 and asialoGM1, which have been shown to undergo strong interactions with ricin. The presence, orientation, and activity of the GSL and antibody films were confirmed using ellipsometry, Fourier transform infrared spectroscopy (FT-IR), and QCM. It was found that the GSLs offered more sensitive detection limits when directly compared with antibodies. Both GSLs had lower detection limits at 5 microg/mL, approximately 5 times lower than were found for antibodies (25 microg/mL), and their linear detection range extended to the highest concentrations tested (100 microg/mL), almost an order of magnitude beyond the saturation point for the antibody sensors. Potential sites for nonspecific adsorption were blocked using serum albumin without sacrificing toxin specificity.     

Peptide aptamers in label-free protein detection: 1. Characterization of the immobilized scaffold

Protein microarray development is absolutely dependent upon the ability to construct interfaces capable of specific, stable, sensitive, and designable recognition of specific proteins. Peptide aptamers, being peptide recognition moieties presented and constrained by a robust scaffold protein, offer one possible solution. The relative uniformity of a scaffold protein across potentially many thousands of arrayed peptide aptamers is predicted to simplify the production of microarrays. This paper describes the generation and assaying characteristics of a scaffold protein adlayer. Orientational control of the scaffold protein STM, a triply mutated form of the stable intracellular protein inhibitor stefin A is achieved with a surface cysteine residue, which leads to the presentation of the scaffold recognition surface to solution. Operational stability of the system is excellent, with only a minor decrease in detection sensitivity over time (less than 1% h-1). We use this system to establish a surface plasmon resonance assay offering a limit of detection of 1 nM (150 ng mL-1) and determine the affinity constant of interaction of STM for a cognate antibody to be KD = 1.47 +/- 0.23 nM. Thus, we have established a solid foundation for the future creation of highly multiplexed peptide aptamer microarrays that will be compatible with a broad range of label-free detection technologies.     

Fluorescence polarization based displacement assay for the determination of small molecules with aptamers

The conversion of an aptamer-target binding event into a detectable signal is an important step in the development of aptamer-based sensors. In this work, we show that the displacement of a fluorescently labeled oligo from the aptamer by the target can be detected by fluorescence polarization (FP). We used Ochratoxin A (OTA), a small organic molecule (MW = 403) as a case study. A detection limit of 5 nM OTA was achieved. The method presented here provides an advantage over fluorophore-quenching systems and other steady-state fluorescence approaches in that no modification of the aptamer or the target is required. Additionally, the signal is produced by the displacement event itself, so no further aggregation or conformational events have to be considered. This analytical method is particularly useful for small targets, as for large targets a direct measurement of the FP change of a labeled aptamer upon binding can be used to determine the concentration of the target. The results presented here demonstrate that aptamers and inexpensive labeled oligos can be used for rapid, sensitive, and specific determination of small molecules by means of FP.     

Thermoresponsive MALDI probe surfaces as a tool for protein on-probe purification

Thin film depositions of rf plasma polymerized N-isopropylacrylamide (ppNIPAM) show a phase transition temperature below which the polymer surface is hydrophilic, and protein nonadsorptive, and above which the polymer surface is hydrophobic, and protein-retentive. Results presented here demonstrate that this thermoresponsive plasma polymer can be coated on the surface of a MALDI probe and subsequently used for on-probe biomolecule cleanup. Specifically, a contaminated biomolecule can be applied to the ppNIPAM coated MALDI probe surface at a temperature above the phase transition temperature, washed using solvent also held above the phase transition temperature, and then analyzed by reducing the probe temperature to room temperature before adding the MALDI matrix. With the use of this approach, it is demonstrated that cytochrome c contaminated with 0.3% SDS, which yields only a very weak MALDI ion signal as directly deposited, can be purified on-probe using the thermoresponsive plasma polymer to improve significantly the ion signal. It is further shown that the decontamination of whole cell protein extracts from cyanobacteria is augmented through the use of the ppNIPAM coated MALDI probe.     

Electron capture dissociation as structural probe for noncovalent gas-phase protein assemblies

Electron capture dissociation (ECD) of proteins in Fourier transform ion cyclotron resonance mass spectrometry usually leads to charge reduction and backbone-bond cleavage, thereby mostly retaining labile, intramolecular noncovalent interactions. In this report, we evaluate ECD of the 84-kDa noncovalent heptameric gp31 complex and compare this with sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD) of the same protein. Unexpectedly, the 21+ charge state of the gp31 oligomer exhibits a main ECD pathway resulting in a hexamer and monomer, disrupting labile, intermolecular noncovalent bonds and leaving the backbone intact. Unexpectedly, the charge separation over the two products is highly proportional to molecular weight. This indicates that a major charge redistribution over the subunits of the complex does not take place during ECD, in contrast to the behavior observed when using SORI-CAD. We speculate that the ejected monomer retains more of its original structure in ECD, when compared to SORI-CAD. ECD of lower charge states of gp31 does not lead to dissociation of noncovalent bonds. We hypothesize that the initial gas-phase structure of the 21+ charge state is significantly different from the lower charge states. These structural differences result in the different reaction pathways when using ECD.     

Surface-modified CdS nanoparticles as a fluorescent probe for the selective detection of cysteine

We present a novel method for the selective detection of cysteine, a sulfur-containing amino acid, which plays a crucial role in many important biological functions such as protein folding. Surface-modified colloidal CdS nanoparticles have been used as a fluorescent probe to selectively detect cysteine in the presence of other amino acids in the micromolar concentration range. Cysteine quenches the emission of CdS in the 0.5-10?μM concentration range, whereas the other amino acids do not affect its emission. Among the other amino acids, histidine is most efficient in quenching the emission of the CdS nanoparticles. The sulfur atom of cysteine plays a crucial role in the quenching process in the 0.5-10?μM concentration range. Cysteine is believed to quench the emission of the CdS nanoparticles by binding to their surface via its negatively charged sulfur atom. This method can potentially be applied for its detection in biological samples.     

Polymeric sequence probe for single DNA detection

A polynucleotide probe, polymeric sequence probe (PSP), was developed for single molecular detections. PSP is a single-stranded DNA molecule with ~2000 tandem repeat target-binding sequences and label-binding sequences. A single PSP can bind to multiple fluorescent complementary oligos to generate a strong fluorescence signal. Single target molecules bound to PSPs can be clearly visualized by a conventional fluorescence microscope. An ultrasensitive PSP-based assay for Mycobacterium tuberculosis was demonstrated.     

Single-molecule measurements of the binding between small molecules and DNA aptamers

Aptamers that bind small molecules can serve as basic biosensing platforms. Evaluation of the binding constant between an aptamer and a small molecule helps to determine the effectiveness of the aptamer-based sensors. Binding constants are often measured by a series of experiments with varying ligand or aptamer concentrations. Such experiments are time-consuming, material nonprudent, and prone to low reproducibility. Here, we use laser tweezers to determine the dissociation constant for aptamer-ligand interactions at the single-molecule level from only one ligand concentration. Using an adenosine 5'-triphosphate disodium salt (ATP) binding aptamer as an example, we have observed that the mechanical stabilities of aptamers bound with ATP are higher than those without a ligand. Comparison of the change in free energy of unfolding (ΔG(unfold)) between these two aptamers yields a ΔG of 33 ± 4 kJ/mol for the binding. By applying a Hess-like cycle at room temperature, we obtained a dissociation constant (K(d)) of 2.0 ± 0.2 μM, a value consistent with the K(d) obtained from our equilibrated capillary electrophoresis (CE) (2.4 ± 0.4 μM) and close to that determined by affinity chromatography in the literature (6 ± 3 μM). We anticipate that our laser tweezers and CE methodologies may be used to more conveniently evaluate the binding between receptors and ligands and also serve as analytical tools for force-based biosensing.     

Spin-stretching of DNA and protein molecules for detection by fluorescence and atomic force microscopy

We have developed a rapid and efficient way of stretching DNA and denatured protein molecules for detection by fluorescence microscopy and atomic force microscopy (AFM). In the described method, a viscous drag created by transient rotational flow stretches randomly coiled DNA molecules or denatured proteins. Stretching is achieved by dispensing a droplet of sample solution containing DNA or denatured protein on a MgCl2-soaked mica surface. We present fluorescent images of straightened lambdaDNA molecules and AFM images of stress-shared, reduced von Willebrand factor as well as straightened lambdaDNA. The described quick and reliable spin-stretching technique will find wide applications in the analysis of single biopolymer molecules.     

Manganese-doped ZnSe quantum dots as a probe for time-resolved fluorescence detection of 5-fluorouracil

Quantum dots (QDs) are generally used for the conventional fluorescence detection. However, it is difficult for the QDs to be applied in time-resolved fluorometry due to their short-lived emission. In this paper, high-quality Mn-doped ZnSe QDs with long-lived emission were prepared using a green and rapid microwave-assisted synthetic approach in aqueous solution. Fluorescence lifetime of the Mn-doped ZnSe QDs was extended as long as 400 μs, which was 10,000 times higher than that of conventional QDs such as CdS, CdSe, and CdTe. The QDs exhibited an excellent photostability over 35 h under continuous irradiation at 260 nm. Capped with mercaptopropionic acid (MPA), the Mn-doped ZnSe QDs were used for the time-resolved fluorescence detection of 5-fluorouracil (5-FU) with the detection limit of 128 nM. The relative standard deviation for seven independent measurements of 1.5 μM 5-FU was 3.8%, and the recovery ranged from 93% to 106%. The results revealed that the Mn-doped ZnSe QDs could be a good candidate as a luminescence probe for highly sensitive time-resolved fluorometry.     

Capillary electrophoresis-based noncompetitive immunoassay for the prion protein using fluorescein-labeled protein A as a fluorescent probe

A novel CE-based noncompetitive immunoassay for prion protein (PrP) was established. Fluorescein isothiocyanate (FITC)-labeled protein A (FITC-PrA) was used as a fluorescent probe to tag monoclonal antibody through noncovalent binding of FITC-PrA to the Fc region of the antibody. The FITC-PrA-Ab was incubated with the analyte, prion protein, under optimized condition, forming the immunocomplex FITC-PrA-Ab-PrP. The complex was separated and analyzed by capillary zone electrophoresis. The addition of carboxymethyl-beta-cyclodextrin in the running buffer as dynamical coating reagent improved the reproducibility and the resolution. The complex was isolated in less than 1 min with theoretical plates of 3.8 x 10(4). Relative standard deviations of peak height and migration time for the complex were 3.46 and 1.48%, respectively. A linear relationship was established for the bovine recombinant prion protein (rPrP) concentration in the range from 0.2 to 2.0 mug/mL and the peak height. The correlation factor was r2 = 0.9969. The estimated detection limit for rPrP was approximately 6 ng/mL, which is 3 times the signal-to-noise ratio. The method was successfully applied for testing blood samples from scrapie-infected sheep.     

Label-free fluorescent detection of ions, proteins, and small molecules using structure-switching aptamers, SYBR Gold, and exonuclease I

We have demonstrated a label-free sensing strategy employing structure-switching aptamers (SSAs), SYBR Gold, and exonuclease I to detect a broad range of targets including inorganic ions, proteins, and small molecules. This nearly universal biosensor approach is based on the observation that SSAs at binding state with their targets, which fold into secondary structures such as quadruplex structure or Y shape structure, show more resistance to nuclease digestion than SSAs at unfolded states. The amount of aptamer left after nuclease reaction is proportional to the concentrations of the targets and in turn is proportional to the fluorescence intensities from SYBR Gold that can only stain nucleic acids but not their digestion products, nucleoside monophosphates (dNMPs). Fluorescent assays employing this mechanism for the detection of potassium ion (K(+)) are sensitive, selective, and convenient. Twenty μM K(+) is readily detected even at the presence of a 500-fold excess of Na(+). Likewise, we have generalized the approach to the specific and convenient detection of proteins (thrombin) and small molecules (cocaine). The assays were then validated by detecting K(+), cocaine, and thrombin in urine and serum or cutting and masking adulterants with good agreements with the true values. Compared to other reported approaches, most limited to G-quadruplex structures, the demonstrated method has less structure requirements of both the SSAs and their complexes with targets, therefore rending its wilder applications for various targets. The detection scheme could be easily modified and extended to detection platforms to further improve the detection sensitivity or for other applications as well as being useful in high-throughput and paralleled analysis of multiple targets.     

One-step homogeneous protein detection based on aptamer probe and fluorescence cross-correlation spectroscopy

A new protein assay based on fluorescence cross-correlation spectroscopy (FCCS) and aptamer probe is developed. In this assay, two spectrally distinct fluorophores labeled aptamer probes are used to recognize and detect thrombin through a sandwich reaction. The sandwich complexes are diffused through a confocal detection volume. The cross-correlation signals can be observed only at the presence of the aptamer probes-protein sandwich complexes. Thrombin is selected as a target to validate the assay. The whole detection process can be completed within an hour with low-nanomolar sensitivity and high specificity. The novel aptamer-based FCCS detection offers a simple, rapid and sensitive method for protein analysis in a homogeneous format.     

Label-free detection of single protein molecules and protein-protein interactions using synthetic nanopores

Nanofabricated pores in 20 nm-thick silicon nitride membranes were used to probe various protein analytes as well as to perform an antigen-antibody binding assay. A two-compartment electrochemical cell was separated by a single nanopore, 28 nm in diameter. Adding proteins to one compartment caused current perturbations in the ion current flowing through the pore. These perturbations correlated with both the charge and the size of the protein or of a protein-protein complex. The potential of this nanotechnology for studying protein-protein interactions is highlighted with the sensitive detection of beta-human chorionic gonadotropin, a hormone and clinical biomarker of pregnancy, by monitoring in real time and at a molecular level the formation of a complex between hormones and antibodies in solution. In this form, the assay compared advantageously to immunoassays, with the important difference that labels, immobilization, or amplification steps were no longer needed. In conclusion, we present proof-of-principle that properties of proteins and their interactions can be investigated in solution using synthetic nanopores and that these interactions can be exploited to measure protein concentrations accurately.     

Label-free detection of single protein molecules using deep UV fluorescence lifetime microscopy

We present the detection of single beta-galactosidase molecules from Escherichia coli (Ecbeta Gal) using deep UV laser-based fluorescence lifetime microscopy. The native fluorescence from intrinsic tryptophan emission has been observed after one-photon excitation at 266 nm. Applying the time-resolved single-photon counting method, we investigated the fluorescence lifetime distribution and the bursts of autofluorescence photons from tryptophan residues in Ecbeta Gal protein as well as fluorescence correlation spectroscopy of Ecbeta Gal. The results demonstrate that deep UV laser-based fluorescence lifetime microscopy is useful for identification of biological macromolecules at the single-molecule level using intrinsic fluorescence.