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.     

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.     

Microfluidic electrochemical aptameric assay integrated on-chip: a potentially convenient sensing platform for the amplified and multiplex analysis of small molecules

Aptamers are artificial oligonucleotides that have been widely employed to design biosensors (i.e., aptasensors). In this work, we report a microfluidic electrochemical aptamer-based sensor (MECAS) by constructing Au-Ag dual-metal array three-electrode on-chip for multiplex detection of small molecules. In combination with the microfluidic channels covering on the glass chip, different targets are transported to the Au electrodes integrated on different positions of the chip. These electrodes are premodified by different kinds of aptamers, respectively, to fabricate different sensing interfaces which can selectively capture the corresponding target. It is an address-dependent sensing platform; thus, with the use of only one electrochemical probe, multitargets can be recognized and detected according to the readout on a corresponding aptamer-modified electrode. In the sensing strategy, the electrochemical probe, [Ru(NH(3))(6)](3+) (RuHex), which can quantitatively bind to surface-confined DNA via electrostatic interaction, was used to produce chronocoulometric signal; Au nanoparticles (AuNPs) were used to improve the sensitivity of the sensor by amplifying the detection signals. Moreover, the sensing interface fabrication, sample incubation, and electrochemical detection were all performed in microfluidic channels. By using this detection chip, we achieved the multianalysis of two model small molecules, ATP, and cocaine, in mixed samples within 40 min. The detection limit of ATP was 3 × 10(-10) M, whereas the detection limit of cocaine was 7 × 10(-8) M. This Au-Ag dual metal electrochemical chip detector integrated MECAS was simple, sensitive, and selective. Also it is similar to a dosimeter which accumulates signal upon exposure. It held promising potential for designing electrochemical devices with high throughput, high automation, and high integration in multianalysis.     

Electrochemical sensing platform based on the highly ordered mesoporous carbon-fullerene system

In this paper, we report a novel all-carbon two-dimensionally ordered nanocomposite electrode system on the basis of the consideration of host-guest chemistry, which utilizes synergistic interactions between a nanostructured matrix of ordered mesoporous carbon (OMC) and an excellent electron acceptor of nanosized fullerene (C 60) to facilitate heterogeneous electron-transfer processes. The integration of OMC-C 60 by covalent interaction, especially its electrochemical applications for electrocatalysis, has not been explored thus far. Such integration may even appear to be counterintuitive because OMC and C 60 provide opposite electrochemical benefits in terms of facilitating heterogeneous electron-transfer processes. Nevertheless, the present work demonstrates the integration of OMC and C 60 can provide a remarkable synergistic augmentation of the current. To illuminate the concept, eight kinds of inorganic and organic electroactive compounds were employed to study the electrochemical response at an OMC-C 60 modified glassy carbon (OMC-C 60/GC) electrode for the first time, which shows more favorable electron-transfer kinetics than OMC/GC, carbon nanotube modified GC, C 60/GC, and GC electrodes. Such electrocatalytic behavior at OMC-C 60/GC electrode could be attributed to the unique physicochemical properties of OMC and C 60, especially the unusual host-guest synergy of OMC-C 60, which induced a substantial decrease in the overvoltage for NADH oxidation compared with GC electrode. The ability of OMC-C 60 to promote electron transfer not only suggests a new platform for the development of dehydrogenase-based bioelectrochemical devices but also indicates a potential of OMC-C 60 to be of a wide range of sensing applications because the electrocatalysis of different electroactive compounds at the OMC-C 60/GC electrode in this work should be a good model for constructing a novel and promising electrochemical sensing platform for further electrochemical detection of other biomolecules.     

Surface-immobilized peptide aptamers as probe molecules for protein detection

We demonstrate the use of surface-immobilized, oriented peptide aptamers for the detection of specific target proteins from complex biological solutions. These peptide aptamers are target-specific peptides expressed within a protein scaffold engineered from the human protease inhibitor stefin A. The scaffold provides stability to the inserted peptides and increases their binding affinity owing to the resulting three-dimensional constraints. A unique cysteine residue was introduced into the protein scaffold to allow orientation-specific surface immobilization of the peptide aptamer and to ensure exposure of the binding site to the target solution. Using dual-polarization interferometry, we demonstrate a strong relationship between binding affinity and aptamer orientation and determine the affinity constant KD for the interaction between an oriented peptide aptamer ST(cys+)_(pep9) and the target protein CDK2. Further, we demonstrate the high selectivity of the peptide aptamer STM_(pep9) by exposing surface-immobilized ST(cys+)_(pep9) to a complex biological solution containing small concentrations of the target protein CDK2.     

A platform for electrochemical sensing of biomolecules based on Europia/reduced graphene oxide nanocomposite

This study aimed at preparing and evaluating the europium oxide–reduced graphene oxide (rGO) composites. Inorganic nanoparticles anchored onto rGO sheets through a facile sonochemical method. The resultant products were characterized by FT-IR, XRD, SEM. Their activity in biomolecules’ analysis were examined by cyclic voltammetry. The rectified electrodes revealed an incredibly electroactive manner. The obtained progress provided excellent materials for scrutiny of biomolecules. The linear relationship was used in the region of 100–1500 µM ascorbic acid (AA), 50–600 µM dopamine (DA), and 10–700 µM uric acid (UA), between current intensities and concentrations. The detection restrictions (LOD) (S/N?=?3) decreased to 8 µM, 1.1 µM and 0.085 µM for AA, DA and UA respectively by differential pulse voltammetry (DPV).     

Noncompetitive phage anti-immunocomplex real-time polymerase chain reaction for sensitive detection of small molecules

Immuno polymerase chain reaction (IPCR) is an analytical technology based on the excellent affinity and specificity of antibodies combined with the powerful signal amplification of polymerase chain reaction (PCR), providing superior sensitivity to classical immunoassays. Here we present a novel type of IPCR termed phage anti-immunocomplex assay real-time PCR (PHAIA-PCR) for the detection of small molecules. Our method utilizes a phage anti-immunocomplex assay (PHAIA) technology in which a short peptide loop displayed on the surface of the M13 bacteriophage binds specifically to the antibody-analyte complex, allowing the noncompetitive detection of small analytes. The phagemid DNA encoding this peptide can be amplified by PCR, and thus, this method eliminates hapten functionalization or bioconjugation of a DNA template while providing improved sensitivity. As a proof of concept, two PHAIA-PCRs were developed for the detection of 3-phenoxybenzoic acid, a major urinary metabolite of some pyrethroid insecticides, and molinate, a herbicide implicated in fish kills. Our results demonstrate that phage DNA can be a versatile material for IPCR development, enabling universal amplification when the common element of the phagemid is targeted or specific amplification when the real time PCR probe is designed to anneal the DNA encoding the peptide. The PHAIA-PCRs proved to be 10-fold more sensitive than conventional PHAIA and significantly faster using magnetic beads for rapid separation of reactants. The assay was validated with both agricultural drain water and human urine samples, showing its robustness for rapid monitoring of human exposure or environmental contamination.     

Surface immobilization of structure-switching DNA aptamers on macroporous sol-gel-derived films for solid-phase biosensing applications

Structure-switching signaling aptamers (ss-aptamers) are single-stranded DNA molecules that are generated through in vitro selection and have the ability to switch between a duplex composed of a quencher-labeled DNA strand (QDNA) hybridized adjacent to a fluorophore label on the aptamer, and an aptamer-target complex wherein the QDNA strand is released, generating a fluorescence signal. While such species have recently emerged as promising biological recognition and signaling elements, very little has been done to evaluate their potential for solid-phase assays. In this study, we demonstrate that high surface area, sol-gel-derived macroporous silica films are suitable platforms for high-density affinity-based immobilization of functional ss-aptamer molecules, allowing for binding of both large and small target analytes with robust signal development. These films are formed using a poly(ethylene glycol) (PEG)-doped sodium silicate material, and we show that it is possible to control the pore size distribution and surface area of the silica film by varying the amount of PEG. Materials with the highest surface area are shown to be able to immobilize up to 6-fold more ss-aptamer than planar glass surfaces, providing greater detection sensitivity and somewhat improved detection limits as compared to immobilization on conventional glass. The solid-phase assay is performed using two different structure-switching signaling aptamers with high selectivity for adenosine 5'-triphosphate and platelet-derived growth factor, respectively, demonstrating that this immobilization scheme should be suitable for a variety of target ligands.     

Robust and highly sensitive fluorescence approach for point-of-care virus detection based on immunomagnetic separation

In this work, robust approach for a highly sensitive point-of-care virus detection was established based on immunomagnetic nanobeads and fluorescent quantum dots (QDs). Taking advantage of immunomagnetic nanobeads functionalized with the monoclonal antibody (mAb) to the surface protein hemagglutinin (HA) of avian influenza virus (AIV) H9N2 subtype, H9N2 viruses were efficiently captured through antibody affinity binding, without pretreatment of samples. The capture kinetics could be fitted well with a first-order bimolecular reaction with a high capturing rate constant k(f) of 4.25 × 10(9) (mol/L)(-1) s(-1), which suggested that the viruses could be quickly captured by the well-dispersed and comparable-size immunomagnetic nanobeads. In order to improve the sensitivity, high-luminance QDs conjugated with streptavidin (QDs-SA) were introduced to this assay through the high affinity biotin-streptavidin system by using the biotinylated mAb in an immuno sandwich mode. We ensured the selective binding of QDs-SA to the available biotin-sites on biotinylated mAb and optimized the conditions to reduce the nonspecific adsorption of QDs-SA to get a limit of detection low up to 60 copies of viruses in 200 μL. This approach is robust for application at the point-of-care due to its very good specificity, precision, and reproducibility with an intra-assay variability of 1.35% and an interassay variability of 3.0%, as well as its high selectivity also demonstrated by analysis of synthetic biological samples with mashed tissues and feces. Moreover, this method has been validated through a double-blind trial with 30 throat swab samples with a coincidence of 96.7% with the expected results.     

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.     

Gold-nanoparticle-embedded polydimethylsiloxane elastomers for highly sensitive Raman detection

A simple, convenient, and efficient method for highly sensitive Raman detection is made by using a Au nanoparticle (AuNP)-embedded polydimethylsiloxane (PDMS) elastomer, referred to as AuNP-PDMS. When this AuNP-PDMS layer is applied to a surface, it can dramatically enhance the Raman signal of detected molecules. Moreover, it can be used for sensitive chemical imaging on solid substrates. As a proof of concept, patterned chemical images of p-aminothiophenol and methylene blue on a Ag substrate are obtained after this chemically patterned Ag substrate is covered by AuNP-PDMS.     

An electrochemical sensing platform based on a new copper complex for the determination of hydrogen peroxide and nitrite

Abstract

A new copper(II) complex [Cu(C₁₂H₂₃N₃)₄Br₂·2H₂O] was synthesized and its structure was characterized by x-ray crystallography and elemental analysis. The copper atom had a distorted octahedron coordination involving two bromide anions and four nitrogen atoms from the 1-decyl-1H-[1,2,4]triazole ligands. Moreover, the electrochemical behavior and electrocatalysis of the carbon paste electrode (Cu-CPE) bulk-modified by the complex have been studied in detail. The Cu-CPE showed excellent electrocatalytic activities toward the reduction of hydrogen peroxide and nitrite, and the detection limit was much lower than that mentioned in earlier reports. This bulk-modified CPE has good reproducibility, long-term stability and surface renewability, which appear promising for constructing chemical sensors.     

Electrochemical detection of quorum sensing signaling molecules by dual signal confirmation at microelectrode arrays

n-Acyl homoserine lactones (AHLs) are produced by gram-negative bacteria to regulate gene expression in a cell density dependent manner. For instance, expression of virulence factors by pathogens such as Pseudomonas aeruginosa is induced only when a threshold concentration of AHLs is reached, which indicates that the bacterial population is big enough to promote infection. In this study, the indicator strain Agrobacterium tumefaciens NTL4 (pZLR4), which carries a β-galactosidase (β-gal) reporter gene under the control of a quorum sensing promoter, was used to develop an electrochemical biosensor to detect AHLs using the model n-(3-oxo)-dodecanoyl-L-homoserine lactone (oxo-C12-HSL), an AHL previously detected in cystic fibrosis patients infected with P. aeruginosa. The substrate 4-aminophenyl β-D-galactopyranoside was used to detect β-gal activity by cyclic voltammetry. Furthermore, simultaneous monitoring of substrate consumption and p-aminophenol production by β-gal allowed on-chip result verification by dual-signal confirmation. The sensor exhibited high reproducibility and accurately detected oxo-C12-HSL in a low picomolar to low nanomolar range in spiked liquid cultures and artificial saliva, as well as AHLs naturally released by P. aeruginosa in culture supernatants. Moreover, detection took just 2 h, required no sample pretreatment or preconcentration steps, and was easier and faster than traditional methods.     

Magnetic bead sensing platform for the detection of proteins

Over the past 5 years, the on-chip detection and manipulation of magnetic beads via magnetoelectronics has emerged as a promising new biosensor platform. Magnetic bead sensing (MBS) provides a highly sensitive and specific technique, enabling these sensors to meet the diagnostic needs that are currently not met by existing technologies. Although many studies have proven the high physical sensitivity of magnetic sensors, the establishment of dose-response curves using MBS is unexplored and their capability to sensitively detect low concentrations of target molecules for diagnostic applications has remained unproven. In this study, we have exploited an alternative MBS concept based on the repositioning of the magnetic beads toward the most sensitive location on the spin valve sensors to allow for highly sensitive immunosensing over a wide range of target concentrations. Furthermore, we present the optimization of the magnetoimmuno assay, i.e., the surface chemistry, the blocking procedure, and the type of magnetic particle, for the highly sensitive and specific detection of S100betabeta, a diagnostic marker for stroke and minor head injury. Finally, a dose-response curve was established that illustrates that our MBS platform can specifically detect S100betabeta down to 27 pg/mL, while maintaining a broad dynamic detection range of approximately 2 decades.     

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.     

Fluorogenic nanocrystals for highly sensitive detection of C-reactive protein

A solid-phase sandwich fluorescence immunoassay using nanocrystals of a fluorogenic precursor, fluorescein diacetate (FDA), conjugated with monoclonal antibodies for the detection of C-reactive protein (CRP), is described. FDA nanocrystals were coated with distearoylglycerophosphoethanolamine (DSPE), modified with amino(poly(ethylene glycol))(PEG(2000)-Amine) as an interface for coupling biomolecules. CRP was chosen as a model analyte because of its widely accepted role as a marker for acute inflammation and prospective heart failure. A low limit of detection (1.10 microg l(-1)) and high precision (CV = 2.72-9.48%) were achieved. Following the immunoreaction, the monoclonal anti-CRP conjugated nanocrystals were released by hydrolysis and dissolution instigated by the addition of a large volume of organic solvent-sodium hydroxide mixture. Using human serum samples from 66 patients with high heart attack risk and 19 healthy blood donors, this CRP fluorescence immunoassay showed a good correlation to the commercially available, turbidimetric immunoassay for CRP. This result was corroborated by the Bland-Altman plot that showed a mean difference between the two methods of only 0.36+/-1.46 mg l(-1). The study demonstrates that the organic fluorogenic FDA nanocrystals can be applied for the detection of CRP, which is a clinically interesting plasma protein with a low limit of detection.     

A nitrite sensor based on a highly sensitive nitrite reductase mediator-coupled amperometric detection

Highly sensitive nitrite sensors have been developed for the first time based on mediator-modified electrodes. Tetraheme cytochrome c nitrite reductase from Sulfurospirillum deleyianum and cytochrome cd(1) nitrite reductase from Paracoccus denitrificans are able to accept electrons from artificial electron donors, which simultaneously act as electron mediators between the enzyme and an amperometric electrode. In addition to methyl viologen, redox-active compounds such as phenazines (phenosafranin, safranin T, N-methylphenazinium, 1-methoxy-N-methylphenazinium) and triarylmethane redox dyes (bromphenol blue and red) were selected from a range of redox compounds exhibiting the most efficient performance for nitrite detection. After precipitation, the electron mediators were incorporated in a graphite electrode material. Enzyme immobilization is performed by entrapment in a poly(carbamoyl sulfonate) (PCS) hydrogel. Diffusion coefficients and apparent heterogeneous rate constants of the mediators as well as homogeneous rate constants of nitrite sensors were determined by chronoamperometry and cyclic voltammetry. The phenosafranin-modified electrode layered with the PCS hydrogel immobilization of tetraheme cytochrome c nitrite reductase yielded linear current responses up to 250 μM nitrite with a sensitivity of 446.5 mA M(-)(1) cm(-)(2). The detection limit of the enzymatic nitrite sensor was found to be 1 μM nitrite.     

Nickle-ion-substituted ceria nanoparticles-based electrochemical sensor for sensitive detection of thiourea

Nickel-substituted ceria nanoparticles (CeO₂:Ni NPs) were prepared by the co-precipitation process under environmental conditions. X-ray diffraction (XRD), field emission-transmission electron microscopy, UV/visible, X-ray photoelectron spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy techniques were successfully used to investigate the crystallographic structure, phase purity, morphology, optical properties, chemical composition, and electrocatalytic properties of the as-prepared ceria NPs. XRD pattern shows the formation of single-phase, highly crystalline, and cubic phase nanostructure with an average of 10 nm crystalline size. As observed from TEM micrographs, particles were highly aggregated may be due to the synthesis in aqueous media. The electrochemical properties and sensing performance of the CeO₂:Ni NPs pasted on glassy carbon electrode were measured against different thiourea concentrations. The fabricated electrode revealed excellent electrocatalytic activity against thiourea concentrations as well as characterization in comparison to the bare electrode. The electrode exhibited a linear detection range between 3.56 and 1000 µM, detection limit 3.56 µM, and sensitivity 2.52 µA mL µM^?1 cm^?2 with regression coefficient 0.995. The electrochemical stability, chemical kinetic, and reproducibility were also examined in the presence of thiourea in phosphate buffer solution.