Fluorescent assay for oxytetracycline based on a long-chain aptamer assembled onto reduced graphene oxide

We report on a fluorescent assay for oxytetracycline (OTC) using a fluorescein-labeled long-chain aptamer assembled onto reduced graphene oxide (rGO). The π-π stacking interaction between aptamer and rGO causes the fluorescence of the label to be almost completely quenched via energy transfer so that the system has very low background fluorescence. The addition of OTC leads to the formation of G-quadruplex OTC complexes and prevents the adsorption of labeled aptamer on the surface of rGO. As a result, fluorescence is restored, and this effect allows for a quantitative assay of OTC over the 0.1–2 μM concentration range and with a detection limit of 10 nM. This method is simple, rapid, selective and sensitive. It may be applied to other small molecule analytes by applying appropriate aptamers.

Bimetallic nano-structured glucose sensing electrode composed of copper atoms deposited on gold nanoparticles

We describe the preparation and sensing capabilities of a bimetallic electrode consisting of copper atoms deposited on gold nanoparticles (GNPs). The electrode was obtained by first constructing a GNP template on the surface of a glassy carbon electrode by exploiting the hydrogen-bonding interactions between pyridine groups on the surface of the GNPs and the carboxy groups of poly(acrylic acid). GNPs (60?nm in diameter) were homogeneously and densely deposited in the template (as revealed by scanning electron microscopy). The electro-deposition of copper ad-atoms on GNPs occurred at an underpotential and was proven by electrochemical techniques. The presence of GNPs in the template accelerated the deposition at low potential due to its beneficial effect on the rate of electron transfer. The new electrode was studied for its response to glucose. Highly stable and reproducible catalytic activity towards glucose oxidation is observed and attributed to the synergistic catalytic effect of the copper atoms on the surface of the GNPs. The detection limit is as low as 50?nM (at a signal-to-noise ratio of 3), and the response is between 200?nM and 10?mM of glucose.

Thermally treated bare gold nanoparticles for colorimetric sensing of copper ions

We demonstrate a sensitive and rapid colorimetric assay for selective detection of copper ions based on the strong coordination between Cu(II) ions and the tetrahydroxyaurate anions [Au(OH)₄]^? on the surface of thermally treated bare gold nanoparticles (GNPs). The method for making the unmodified GNPs is simple and results in a nanomaterial with a highly specific response to Cu(II). The thermal treatment of the bare GNPs and the recognition of Cu(II) ions is accomplished in a single step within 5 min. The presence of Cu(II) causes the color to change from red to purple-blue. The limit of detection (LOD) is 0.04 μM of Cu(II) when using UV–vis spectrometry and ratioing the absorbances at 650 and 515 nm, respectively. The method also is amenable to bare eye (visual) inspection and in this case has an LOD of 2.0 μM of Cu(II).

Binding kinetics of human cellular prion detection by DNA aptamers immobilized on a conducting polypyrrole

We developed a biosensor based on the surface plasmon resonance (SPR) method for the study of the binding kinetics and detection of human cellular prions (PrP^C) using DNA aptamers as bioreceptors. The biosensor was formed by immobilization of various biotinylated DNA aptamers on a surface of conducting polypyrrole modified by streptavidin. We demonstrated that PrP^C interaction with DNA aptamers could be followed by measuring the variation of the resonance angle. This was studied using DNA aptamers of various configurations, including conventional single-stranded aptamers that contained a rigid double-stranded supporting part and aptamer dimers containing two binding sites. The kinetic constants determined by the SPR method suggest strong interaction of PrP^C with various DNA aptamers depending on their configuration. SPR aptasensors have a high selectivity to PrP^C and were regenerable by a brief wash in 0.1 M NaOH. The best limit of detection (4 nM) has been achieved with this biosensor based on DNA aptamers with one binding site but containing a double-stranded supporting part.

Sandwich immunoassay for alpha-fetoprotein in human sera using gold nanoparticle and magnetic bead labels along with resonance Rayleigh scattering readout

We describe a sensitive sandwich immunoassay for alpha-fetoprotein (AFP). It is making use of gold nanoparticles (GNPs) and magnetic beads (MBs) as labels, and of resonance Rayleigh scattering for detection. Two antibodies were labeled with GNPs and MBs, respectively, and MB-antigen-GNP complexes were formed in the presence of antigens. The MB labels also serve as solid phase carriers that can be used to magnetically separate the immuno complex. The GNP labels are used as optical probes, and Rayleigh scattering was used to determine the concentration of free GNPs-antibody after separation of the MB-antigen-GNP complexes. The concentration of AFP is related to the intensity of light scattered by free GNPs in the 13.6 pM to 436 pM concentration range, and the limit of detection is 13.6 pM. The method was applied to the determination of AFP in sera of cancer patients, and the results agree well with those obtained by conventional ELISA.

Fluorogenic assays for activated protein C using aptamer modified magnetic beads

We describe a fluorogenic assay for activated protein C (APC) by using magnetic beads modified with DNA aptamers, taking advantage of strong binding affinity of aptamer, facile magnetic separation, and signal amplification via an enzymatic reaction. APC is specifically captured from a sample by the DNA aptamers on magnetic beads, and the concentrated APC then catalyzes the conversion of a fluorogenic substrate of APC to a fluorescent product. Detection of APC is achieved by measuring the generated product. This method is simple, sensitive, and specific. APC can be detected at 0.4 pM concentration level in a sample volume of 250 μL, corresponding to 0.1 femtomole of APC, when 2-h enzymatic reaction is employed. The proteins thrombin, trypsin, proteinase K, chymotrypsin, and elastase do not interfere.

Label-free aptasensor for thrombin using a glassy carbon electrode modified with a graphene-porphyrin composite

We report on an electrochemical aptasensor for the ultrasensitive determination of thrombin. A glassy carbon electrode modified with a graphene-porphyrin nanocomposite exhibits excellent electrochemical activity and can be used as a redox probe in differential pulse voltammetry of the porphyrin on its surface. The thrombin aptamer is then immobilized via p-stacking interactions between aptamer and graphene and π-π stacking with porphyrin simultaneously. The resulting electrochemical aptasensor displays a linear response to thrombin in the 5–1,500 nM concentration range and with a limit of detection of 0.2 nM (at an S/N of 3). The sensor benefits from the synergetic effects of graphene (with its high conductivity and high surface area), of the porphyrin (possessing excellent electrochemical activity), and of the aptamer (with its high affinity and specificity). This kind of aptasensor conceivably represents a promising tool for bioanalytical applications.

Detection of thrombin using an excimer aptamer switch labeled with dual pyrene molecules

We constructed an excimer aptamer probe containing one pyrene molecule at each end of a DNA aptamer to achieve the detection of thrombin, which binds to the heparin-binding site of thrombin with high binding affinity. The specific binding of thrombin to the excimer aptamer probe brought the two pyrene molecules at the termini of the duplex of the aptamer into close proximity, generating an excimer. The excimer emitted a distinct fluorescence peak, and fluorometric measurement of excimer allowed the sensitive detection of thrombin. The effects of experimental conditions like pH, ionic strength, and cations were investigated and optimized. The detection limit for thrombin was about 42 pM. This aptamer switch has potential in the study of molecular interactions and protein sensing with other switch-based detection strategy.

Templated in-situ synthesis of gold nanoclusters conjugated to drug target bacterial enoyl-ACP reductase,and their application to the detection of mercury ions using a test stripe

Fluorescent gold nanoclusters (AuNCs) were synthesized using a drug target bacterial enoyl-ACP reductase (FabI) as a template. The physical and chemical properties of the AuNCs were studied by UV-vis absorption, fluorescence, X-ray photoelectron spectroscopy and TEM. The AuNCs-FabI conjugate was prepared by in situ reduction of tetrachloroaurate in the presence of FabI. The conjugated particles were loaded onto nylon membranes by taking advantage of the electrostatic interaction between the negatively charged AuNCs@FabI and the nylon film which is positively charged at pH 7.4. This results in the formation of a test stripe with sensor spots that can be used to detect Hg(II) ion in the 1 nM to 10 μM concentration range. The test stripes are simple, convenient, selective, sensitive, and can be quickly read out with bare eyes after illumination with a UV lamp.

Simple optical determination of silver ion in aqueous solutions using benzo crown-ether modified gold nanoparticles

We describe a method for the modification of gold nanoparticles (Au-NPs) with benzo-15-crown-5 that led to the development of a colorimetric assay for Ag(I) ion. The brown color of a solution of the modified Au-NPs turns to purple on addition of Ag(I) ion. The ratio of the UV–vis absorption at 600 nm and 525 nm is proportional to the concentration of Ag(I) ions in the range from 20 to 950 nM, and the detection limit is 12.5 nM. Other metal ions do not interfere if present in up to millimolar concentrations. The method enables a rapid determination of Ag(I) in lake and drinking water and is amenable to bare-eye readout.

Water-soluble homo-oligonucleotide stabilized fluorescent silver nanoclusters as fluorescent probes for mercury ion

Water–soluble fluorescent silver nanoclusters (Ag NCs) were prepared with the assistance of commercially available polyinosinic acid (PI) or polycytidylic acid (PC). The fluorescence of the Ag NCs is effectively quenched by trace mercury(II) ions, which can be applied for their detection. The response of the Ag NCs prepared with PI to Hg(II) ion is linear in the Hg(II) concentration range from 0.05 to 1.0 μM (R²?=?0.9873), and from 0.5 to 10 μM of Hg(II) (R²?=?0.9971) for Ag NCs prepared with PC. The detection limits are 3.0 nM and 9.0 nM (at an S/N of 3), respectively. The method is simple, sensitive and fairly selective.

Strong enhancement of the chemiluminescence of the cerium(IV)-thiosulfate reaction by carbon dots,and its application to the sensitive determination of dopamine

We show that the very weak chemiluminescence (CL) of the Ce(IV)-thiosulfate system is enhanced by a factor of ~150 in the presence of fluorescent carbon dots (C-dots). The C-dots were prepared by a solvothermal method and characterized by fluorescence spectra and transmission electron microscopy. Possible mechanisms that lead to the effect were elucidated by recording fluorescence and CL spectra. It is found that dopamine at even nanomolar levels exerts a diminishing effect on the enhancement of CL. This was exploited to design a method for the determination of dopamine in the concentration range from 2.5 nM to 20 μM, with a limit of detection (at 3 s) of 1.0 nM. Dopamine was determined by this method in spiked human plasma samples with satisfactory results.

Improving surface plasmon resonance imaging of DNA by creating new gold and silver based surface nanostructures

The use of nanoparticles (NPs) can substantially improve the analytical performance of surface plasmon resonance imaging (SPRi) in general, and in DNA sensing in particular. In this work, we report on the modification of the gold surface of commercial biochips with gold nanospheres, silica-coated gold nanoshells, and silver nanoprisms, respectively. The NPs were tethered onto the surface of the chip and functionalized with a DNA probe. The effects of tethering conditions and varying nanostructures on the SPRi signals were evaluated via hybridization assays. The results showed that coupling between planar surface plasmons and electric fields, generated by localized surface plasmons of the NPs, is mandatory for signal enhancement. Silver nanoprisms gave the best results in improving the signal change at a target DNA concentration of <50 nM by +50 % (compared to a conventional SPRi chip). The limit of detection for the target DNA was 0.5 nM which is 5 times less than in conventional SPRi.

Chronocoulometric DNA biosensor based on a glassy carbon electrode modified with gold nanoparticles, poly(dopamine) and carbon nanotubes

We describe a sensitive chronocoulometric biosensor for the sequence-specific detection of DNA. It is based on a glassy carbon electrode modified with multi-walled carbon nanotubes, polydopamine, and gold nanoparticles. The ruthenium(III)hexammine complex acts as the electrochemical indicator. Electrochemical impedance spectra and scanning electron microscopy are employed to investigate the assembly of the electrode surface. The signals of the ruthenium complex electrostatically bound to the anionic phospho groups of the DNA strands are measured by chronocoulometry before and after hybridization. The difference in signal intensity is linearly related to the logarithm of the concentration of the target DNA in the range of 1.0 nM to 10 fM with a detection limit of 3.5fM (S/N?=?3) under optimal conditions. This biosensor exhibits excellent sensitivity and selectivity and has been used for an assay of complementary target DNA in human serum sample with satisfactory results.

Highly sensitive and selective voltammetric detection of mercury(II) using an ITO electrode modified with 5-methyl-2-thiouracil, graphene oxide and gold nanoparticles

We have developed an electrochemical sensor for highly selective and sensitive determination of Hg(II). It is based on the specific binding of 5-methyl-2-thiouracil (MTU) and Hg(II) to the surface of an indium tin oxide (ITO) electrode modified with a composite made from graphene oxide (GO) and gold nanoparticles (AuNPs). This leads to a largely enhanced differential pulse voltammetric response for Hg(II). Following optimization of the method, a good linear relationship (R?=?0.9920) is found between peak current and the concentration of Hg(II) in the 5.0–110.0 nM range. The limit of detection (LOD) is 0.78 nM at a signal-to-noise ratio of 3. A study on the interference by several metal ions revealed no interferences. The feasibility of this method was demonstrated by the analyses of real water samples. The LODs are 6.9, 1.0 and 1.9 nM for tap water, bottled water and lake water samples, respectively, and recoveries for the water samples spiked with 8.0, 50.0 and 100.0 nM were 83.9–96.8 %, with relative standard deviations ranging from 3.3 % to 5.2 %.

Mass-amplifying quantum dots in a fluorescence polarization-based aptasensor for ATP

We report on a fluorescence polarization assay for the detection of the target analyte ATP by making use of an aptasensor and of mass-amplifying CdTe-CdS quantum dots. The ATP aptamer was modified with digoxin antigen and hybridized with its complementary DNA that was modified with the CdTe-CdS quantum dots. Following the addition of digoxin antibody, the mass-amplifying aptasensor probe is formed as a result of the immuno reaction. In the presence of ATP, the polarization of fluorescence decreases because the digoxin antibody becomes dissociated due to the recognition of the ATP by the ATP aptamer. Under optimized conditions, the method has a linear response to ATP in the 10 to 350 μM concentration range, and the limit of detection is 3.7 μM. The method combines the specific recognition capability of aptamers with the sensitivity of an immunoreaction. It has good selectivity and sensitivity, and can be used to detect ATP in serum samples.

Selection and identification of ssDNA aptamers recognizing zearalenone

Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin produced by Fusarium graminearum on maize and barley. Because most current methods of ZEN detection rely on the use of low-stability antibodies or expensive equipment, we sought to develop a rapid, low-cost determination method using aptamers instead of antibodies as the specific recognition ligands. This work describes the isolation and identification of single-stranded DNA (ssDNA) aptamers recognizing ZEN using the modified systematic evolution of ligands by exponential enrichment methodology based on magnetic beads. After 14 rounds of repeated selection, a highly enriched ssDNA library was sequenced and 12 representative sequences were assayed for their affinity and specificity. The best aptamer, 8Z₃₁, with a dissociation constant (K _d) of 41?±?5 nM, was successfully applied in the specific detection of ZEN in binding buffer and in real samples based on a magnetic separation/preconcentration procedure. This analytical method provided a linear range from 3.14?×?10^?9 to 3.14?×?10^?5 M for ZEN, and the detection limit was 7.85?×?10^?10 M. The selected aptamers are expected to be used in the potential development of affinity columns, biosensors, or other analytical systems for the determination of ZEN in food and agricultural products.

A Surface Enhanced Raman Scattering (SERS) microdroplet detector for trace levels of crystal violet

We report on a microfluidic platform that integrates a winding microdroplet chip and a surface-enhanced Raman scattering (SERS) detection system for trace determination of crystal violet (CV). Colloidal silver was applied to generate SERS. Compared to the continuous flow microfluidic system, the microdroplet based detection described here effectively eliminates any memory effects. Effects of flow pattern, droplet size, surfactant, and position of detection were optimized. Under optimal conditions, there is a linear correlation between signal and the concentration of CV in the 10 nM to 800 nM range, with a correlation coefficient (R²) of 0.9967. The limit of detection in water is 3.6 nM.

A lanthanide nanoparticle-based luminescent probe for folic acid

We report on a novel luminescent method for the detection of folic acid (FA), a member of the vitamin B family. Y₂O₃ nanoparticles were doped with europium(III) ions and surface-modified with captopril. Their fluorescence is quenched by FA, and intensity is a function of folic acid concentration in the 0.1 – 40 μM concentration range. The detection limit is 83 nM of FA at pH 7 and room temperature.