DNA Sensors and Aptasensors Based on the Hemin/G‐quadruplex‐Controlled Aggregation of Au NPs in the Presence of L‐Cysteine

L‐cysteine induces the aggregation of Au nanoparticles (NPs), resulting in a color transition from red to blue due to interparticle plasmonic coupling in the aggregated structure. The hemin/G‐quadruplex horseradish peroxidase‐mimicking DNAzyme catalyzes the aerobic oxidation of L‐cysteine to cystine, a process that inhibits the aggregation of the NPs. The degree of inhibition of the aggregation process is controlled by the concentration of the DNAzyme in the system. These functions are implemented to develop sensing platforms for the detection of a target DNA, for the analysis of aptamer‐substrate complexes, and for the analysis of L‐cysteine in human urine samples. A hairpin DNA structure that includes a recognition site for the DNA analyte and a caged G‐quadruplex sequence, is opened in the presence of the target DNA. The resulting self‐assembled hemin/G‐quadruplex acts as catalyst that controls the aggregation of the Au NPs. Also, the thrombin‐binding aptamer folds into a G‐quadruplex nanostructure upon binding to thrombin. The association of hemin to the resulting G‐quadruplex aptamer‐thrombin complex leads to a catalytic label that controls the L‐cysteine‐mediated aggregation of the Au NPs. The hemin/G‐qaudruplex‐controlled aggregation of Au NPs process is further implemented for visual and spectroscopic detection of L‐cysteine concentration in urine samples.     

Fluorescence detection of lead(II) ions through their induced catalytic activity of DNAzymes

We have developed a fluorescence approach for the highly selective and sensitive detection of Pb(2+) ions using AGRO100, a G-quadruplex DNAzyme. The sensing strategy is based on Pb(2+) ions inducing increased DNAzyme activity of AGRO100 in the presence of hemin, which acts as a cofactor to catalyze H(2)O(2)-mediated oxidation of Amplex UltraRed (AUR). A test of eight aptamers of various sequences for the detection of Pb(2+) ions revealed that AGRO100 performed the best in terms of sensitivity. The AGRO100-AUR probe exhibited high selectivity (>100-fold) toward Pb(2+) ions over other tested metal ions. The fluorescence intensity (excitation/emission maxima, ca. 561/592 nm) of the AUR product was proportional to the concentration of Pb(2+) ions over the range 0-1000 nM, with a linear correlation (R(2) = 0.98). For 5 mM Tris-acetate (pH 7.4) solutions in the presence and absence of 100 mM NaCl, the AGRO100-AUR probe provided limits of detection (signal-to-noise ratio = 3) for Pb(2+) ions of 1.0 and 0.4 nM, respectively. We validated the practicality of the use of the AGRO100-AUR probe for the determination of the concentrations of Pb(2+) ions in soil samples. This approach allows the determination of the concentrations of Pb(2+) ions with simplicity, selectivity, and sensitivity.     

Electrochemical DNAzyme sensor for lead based on amplification of DNA-Au bio-bar codes

An electrochemical DNAzyme sensor for sensitive and selective detection of lead ion (Pb(2+)) has been developed, taking advantage of catalytic reactions of a DNAzyme upon its binding to Pb(2+) and the use of DNA-Au bio-bar codes to achieve signal enhancement. A specific DNAzyme for Pb(2+) is immobilized onto an Au electrode surface via a thiol-Au interaction. The DNAzyme hybridizes to a specially designed complementary substrate strand that has an overhang, which in turn hybridizes to the DNA-Au bio-bar code (short oligonucleotides attached to 13 nm gold nanoparticles). A redox mediator, Ru(NH3)6(3+), which can bind to the anionic phosphate of DNA through electrostatic interactions, serves as the electrochemical signal transducer. Upon binding of Pb(2+) to the DNAzyme, the DNAzyme catalyzes the hydrolytic cleavage of the substrate, resulting in the removal of the substrate strand along with the DNA bio-bar code and the bound Ru(NH3)6(3+) from the Au electrode surface. The release of Ru(NH3)6(3+) results in lower electrochemical signal of Ru(NH3)6(3+) confined on the electrode surface. Differential pulse voltammetry (DPV) signals of Ru(NH3)6(3+) provides quantitative measures of the concentrations of Pb(2+), with a linear calibration ranging from 5 nM to 0.1 microM. Because each nanoparticle carries a large number of DNA strands that bind to the signal transducer molecule Ru(NH3)6(3+), the use of DNA-Au bio-bar codes enhances the detection sensitivity by five times, enabling the detection of Pb(2+) at a very low level (1 nM). The DPV signal response of the DNAzyme sensor is negligible for other divalent metal ions, indicating that the sensor is highly selective for Pb(2+). Although this DNAzyme sensor is demonstrated for the detection of Pb(2+), it has the potential to serve as a general platform for design sensors for other small molecules and heavy metal ions.     

Graphene-DNAzyme based biosensor for amplified fluorescence "turn-on" detection of Pb2+ with a high selectivity

On the basis of the remarkable difference in affinity of graphene (GO) with ssDNA containing a different number of bases in length, we for the first time report a GO-DNAzyme based biosensor for amplified fluorescence "turn-on" detection of Pb(2+). A FAM-labeled DNAzyme-substrate hybrid acted as both a molecular recognition module and signal reporter and GO as a superquencher. By taking advantage of the super fluorescence quenching efficiency of GO, our proposed biosensor exhibits a high sensitivity toward the target with a detection limit of 300 pM for Pb(2+), which is lower than previously reported for catalytic beacons. Moreover, with the choice of a classic Pb(2+)-dependent GR-5 DNAzyme instead of 8-17 DNAzyme as the catalytic unit, the newly designed sensing system also shows an obviously improved selectivity than previously reported methods. Moreover, the sensing system was used for the determination of Pb(2+) in river water samples with satisfying results.     

DNAzyme-based colorimetric sensing of lead (Pb(2+)) using unmodified gold nanoparticle probes

Novel functional oligonucleotides, especially DNAzymes with RNA-cleavage activity, have been intensively studied due to their potential applications in therapeutics and sensors. Taking advantage of the high specificity of 17E DNAzyme for Pb(2+), highly sensitive and selective fluorescent, electrochemical and colorimetric sensors have been developed for Pb(2+). In this work, we report a simple, sensitive and label-free 17E DNAzyme-based sensor for Pb(2+) detection using unmodified gold nanoparticles (GNPs) based on the fact that unfolded single-stranded DNA could be adsorbed on the citrate protected GNPs while double-stranded DNA could not. By our method the substrate cleavage by the 17E DNAzyme in the presence of Pb(2+) could be monitored by color change of GNPs, thereby Pb(2+) detection was realized. The detection of Pb(2+) could be realized within 20?min, with a detection limit of 500?nM. The selectivity of our sensor has been investigated by challenging the sensing system with other divalent metal ions. Since common steps such as modification and separation could be successfully avoided, the sensor developed here could provide a simple, cost-effective yet rapid and sensitive measurement tool for Pb(2+) detection and may prove useful in the development of sensors for clinical toxicology and environmental monitoring in the future.     

Colorimetric detection of trace copper ions based on catalytic leaching of silver-coated gold nanoparticles

A colorimetric, label-free, and nonaggregation-based silver coated gold nanoparticles (Ag/Au NPs) probe has been developed for detection of trace Cu(2+) in aqueous solution, based on the fact that Cu(2+) can accelerate the leaching rate of Ag/Au NPs by thiosulfate (S(2)O(3)(2-)). The leaching of Ag/Au NPs would lead to dramatic decrease in the surface plasmon resonance (SPR) absorption as the size of Ag/Au NPs decreased. This colorimetric strategy based on size-dependence of nanoparticles during their leaching process provided a highly sensitive (1.0 nM) and selective detection toward Cu(2+), with a wide linear detection range (5-800 nM) over nearly 3 orders of magnitude. The cost-effective probe allows rapid and sensitive detection of trace Cu(2+) ions in water samples, indicating its potential applicability for the determination of copper in real samples.     

Impedimetric immobilized DNA-based sensor for simultaneous detection of Pb2+, Ag+, and Hg2+

An unlabeled immobilized DNA-based sensor was reported for simultaneous detection of Pb(2+), Ag(+), and Hg(2+) by electrochemical impedance spectroscopy (EIS) with [Fe(CN)(6)](4-/3-) as redox probe, which consisted of three interaction sections: Pb(2+) interaction with G-rich DNA strands to form G-quadruplex, Ag(+) interaction with C-C mismatch to form C-Ag(+)-C complex, and Hg(2+) interaction with T-T mismatch to form T-Hg(2+)-T complex. Circular dichroism (CD) and UV-vis spectra indicated that the interactions between DNA and Pb(2+), Ag(+), or Hg(2+) occurred. Upon DNA interaction with Pb(2+), Ag(+), and Hg(2+), respectively, a decreased charge transfer resistance (R(CT)) was obtained. Taking advantage of the R(CT) difference (ΔR(CT)), Pb(2+), Ag(+), and Hg(2+) were selectively detected with the detection limit of 10 pM, 10 nM, and 0.1 nM, respectively. To simultaneously (or parallel) detect the three metal ions coexisting in a sample, EDTA was applied to mask Pb(2+) and Hg(2+) for detecting Ag(+); cysteine was applied to mask Ag(+) and Hg(2+) for detecting Pb(2+), and the mixture of G-rich and C-rich DNA strands were applied to mask Pb(2+) and Ag(+) for detecting Hg(2+). Finally, the simple and cost-effective sensor could be successfully applied for simultaneously detecting Pb(2+), Ag(+), and Hg(2+) in calf serum and lake water.     

Colorimetric assay of lead ions in biological samples using a nanogold-based membrane

We have developed a simple paper-based colorimetric membrane for sensing lead ions (Pb(2+)) in aqueous solutions. The nitrocellulose membrane (NCM) was used to trap bovine serum albumin (BSA) modified 13.3-nm Au nanoparticles (BSA-Au NPs), leading to the preparation of a nanocomposite film of a BSA-Au NP-decorated membrane (BSA-Au NPs/NCM). The BSA-Au NPs/NCM operates on the principle that Pb(2+) ions accelerate the rate of leaching of Au NPs induced by thiosulfate (S(2)O(3)(2-)) and 2-mercaptoethanol (2-ME). The BSA-Au NPs/NCM allowed for the detection of Pb(2+) by the naked eye in nanomolar aqueous solutions in the presence of leaching agents such as S(2)O(3)(2-) and 2-ME. We employed the assistance of microwave irradiation to shorten the reaction time (<10 min) for leaching the Au NPs. Under optimal solution conditions (5 mM glycine-NaOH (pH 10), S(2)O(3)(2-) (100 mM), and 2-ME (250 mM), microwaves (450 W)), the BSA-Au NPs/NCM allowed the detection of Pb(2+) at concentrations as low as 50 pM with high selectivity (at least 100-fold over other metal ions). This cost-effective sensing system allowed for the rapid and simple determination of the concentrations of Pb(2+) ions in real samples (in this case, sea water, urine, and blood samples).     

Molecularly imprinted Au nanoparticles composites on Au surfaces for the surface plasmon resonance detection of pentaerythritol tetranitrate, nitroglycerin, and ethylene glycol dinitrate

Molecularly imprinted Au nanoparticles (NPs) composites are generated on Au-coated glass surfaces. The imprinting process involves the electropolymerization of thioaniline-functionalized Au NPs (3.5 nm) on a thioaniline monolayer-modified Au surface in the presence of a carboxylic acid, acting as a template analogue for the respective explosive. The exclusion of the imprinting template from the Au NPs matrix yields the respective imprinted composites. The binding of the analyte explosives to the Au NPs matrixes is probed by surface plasmon resonance spectroscopy, SPR, where the electronic coupling between the localized plasmon of the Au NPs and the surface plasmon wave leads to the amplification of the SPR responses originating from the dielectric changes of the matrixes upon binding of the different explosive materials. The resulting imprinted matrixes reveal high affinities and selectivity toward the imprinted explosives. Using citric acid as an imprinting template, Au NPs matrixes for the specific analysis of pentaerythritol tetranitrate (PETN) or of nitroglycerin (NG) were prepared, leading to detection limits of 200 fM and 20 pM, respectively. Similarly, using maleic acid or fumaric acid as imprinting templates, high-affinity sensing composites for ethylene glycol dinitrate (EGDN) were synthesized, leading to a detection limit of 400 fM for both matrixes.     

Amplified chemiluminescence surface detection of DNA and telomerase activity using catalytic nucleic acid labels

A G-rich nucleic acid sequence binds hemin and yields a biocatalytic complex (DNAzyme) of peroxidase activity, namely, the biocatalyzed generation of chemiluminescence in the presence of H(2)O(2) and luminol. The DNAzyme is used as a label for the amplified detection of DNA, or for the analysis of telomerase activity in cancer cells, using chemiluminescence as an output signal. In one configuration, the analyzed DNA is hybridized with a primer nucleic acid that is associated with a Au surface, and the DNAzyme label is hybridized with the surface-confined analyte DNA. The DNA is analyzed with a detection limit of approximately 1 x 10(-)(9) M. In the second system, telomerase from HeLa cancer cells induces telomerization of a primer associated with a Au surface and the complementary DNAzyme units are hybridized with the telomere to yield the chemiluminescence. The detection limit of the system corresponds to 1000 HeLa cells in the analyzed sample.     

Immobilization of a catalytic DNA molecular beacon on Au for Pb(II) detection

A Pb(II)-specific DNAzyme fluorescent sensor has been modified with a thiol moiety in order to immobilize it on a Au surface. Self-assembly of the DNAzyme is accomplished by first adsorbing the single-thiolated enzyme strand (HS-17E-Dy) followed by adsorption of mercaptohexanol, which serves to displace any Au-N interactions and ensure that DNA is bound only through the S-headgroup. The preformed self-assembled monolayer is then hybridized with the complementary fluorophore-containing substrate strand (17DS-Fl). Upon reaction with Pb(II), the substrate strand is cleaved, releasing a fluorescent fragment for detection. Fluorescence intensity may be correlated with original Pb(II) concentration, and a linear calibration was obtained over nearly four decades: 10 microM > or = [Pb(II)] > or = 1 nM. The immobilized DNAzyme is a robust system; it may be regenerated after cleavage, allowing multiple sensing cycles. In addition, drying of fully assembled DNAzyme before reaction with Pb(II) does not significantly affect analytical performance. These results demonstrate that, in comparison with solution-based schemes, immobilization of the DNAzyme sensor onto a Au surface lowers the detection limit (from 10 to 1 nM), maintains activity and specificity, and allows sensor regeneration and long-term storage. Realization of Pb(II) detection through an immobilized DNAzyme is the first important step toward creation of a stand-alone, portable Pb(II) detection device such as those immobilizing DNAzyme recognition motifs in the nanofluidic pores of a microfluidic-nanofluidic hybrid multilayer device.     

Ultrasensitive Pb2+ detection by glutathione-capped quantum dots

Water-soluble and stable quantum dots (QDs), CdTe and CdZnSe, are applied for ultrasensitive Pb(2+) detection. These QDs are capped with glutathione (GSH) shells. GSH and its polymeric form, phytochelatin, are employed by nature to detoxify heavy metal ions. As a result of specific interaction, the fluorescence intensity of GSH-capped QDs is selectively reduced in the presence of heavy metal ions such as Pb(2+). The detection limit of Pb(2+) is found to be 20 nM due to the superior fluorescence properties of QDs. Detailed studies by spectroscopy, microscopy, and dynamic light scattering show that competitive GSH binding of Pb(2+) with the QD core changed both the surface and photophysical properties of the QDs. Fluorescence of QDs is quenched, and QD aggregation occurs. Coupling the GSH-capped QDs with a high-throughput detection system, we have developed a simple scheme for quick and ultrasensitive Pb(2+) detection without the need for additional electronic devices. In the presence of ionic mixtures, our system is still capable of Pb(2+) detection with a detection limit as low as 40 nM. The system only becomes less sensitive when the ionic mixture is present at a very high concentration (i.e., > or =50 microM).     

Computer-Readable DNAzyme Assay on Disc for ppb-Level Lead Detection

A method for the convenient detection of lead at the parts-per-billion (ppb)-level has been developed; it uses a conventional compact disc (CD) as the platform for preparing DNAzyme assays and an unmodified optical drive of ordinary desktop/laptop computers as the readout device. In particular, by immobilization of Pb(2+)-specific DNAzyme sensing constructs on the "transparent side" of a conventional CD-R via mild surface reactions, the Pb(2+) concentration can be determined by a free diagnostic program that checks the error distribution on the CD (i.e., it extracts the number of errors in a prerecorded audio file). The reading errors increase monotonically over a wide range of Pb(2+) concentrations (from 10 nM to 1 mM), and the selectivity is confirmed by testing several other divalent cations (Zn(2+), Ba(2+), Mg(2+), Ca(2+), Cu(2+), and Hg(2+)).     

"Turn-on" chemiluminescence sensor for the highly selective and ultrasensitive detection of Hg2+ ions based on interstrand cooperative coordination and catalytic formation of gold nanoparticles

Monitoring the levels of potentially toxic metal ions such as Hg(2+) in aquatic ecosystems is important because this ion can have severe effects on human health and the environment. Thus, a novel chemiluminescence (CL) sensor is developed for the highly selective and ultrasensitive detection of Hg(2+) ions in aqueous solution, based on thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination chemistry and subsequent formation of gold nanoparticles (Au NPs) in a HAuCl(4)/NH(2)OH reaction. The thus-formed Au NPs trigger the reaction between luminol and AgNO(3), producing CL emission. This novel CL technique has several advantages including high sensitivity (0.01 ppb) and selectivity over a spectrum of interfering metal ions. In view of these advantages, as well as the cost-effective, minimized working steps and portable features of the CL techniques, we expect that this CL sensor will be a promising candidate for the field detection of toxic Hg(2+) ions in environment, water, and food samples.     

Bimetallic chips for a surface plasmon resonance instrument

In this work we have replaced a commonly used Au film with an Au/Ag bimetallic film working as the sensing chip in the commercial surface plasmon resonance (SPR) instrument, Biacore 3000. The bimetallic chip gives out sharper SPR dip than that by the chip made of an Au film. The applicability and stability of the bimetallic chip on the Biacore 3000 are tested by real-time monitoring of biological coupling processes between sulfamethoxazole (SMX) and anti-SMX, which is useful in rapid detection of contaminations in food products. The bimetallic chip performs stably in 6 times' usage and regeneration. Our experiment is helpful to push the bimetallic film into real applications in commercial SPR instruments.     

Effect of surface plasmon resonance on the photocatalytic activity of Au/TiO2 under UV/visible illumination

In this study, gold-loaded titanium dioxide was prepared by an impregnation method to investigate the effect of surface plasmon resonance (SPR) on photoactivity. The deposited gold nanoparticles (NPs) absorb visible light because of SPR. The effects of both the gold content and the TiO2 size of Au/TiO2 on SPR and the photocatalytic efficiency were investigated. The morphology, crystal structure, light absorption, emission from the recombination of a photoexcited electron and hole, and the degree of aggregation were investigated using transmission electron microscopy (TEM), X-ray diffraction (XRD), UV-visible-diffuse reflectance spectra (UV-VIS-DRS), photoluminescence (PL) spectroscopy, and turbidimetry, respectively. Photocatalytic activity was evaluated by the decolorization of methyl orange solution over modified titania under UV and UV/GLED (green light emitting diode) illumination. Au/TiO2 NPs exhibited an absorption peak (530-570 nm) because of SPR. The results of our photocatalytic experiments indicated that the UV-inducedly photocatalytic reaction rate was improved by simultaneously using UV and green light illumination; this corresponds to the adsorption region of SPR. Au/TiO2 could use the enhanced electric field amplitude on the surface of the Au particle in the spectral vicinity of its plasmon resonance and thus improve the photoactivity. Experimental results show that the synergistic effect between UV and green light for the improvement of photoactivity increases with increasing the SPR absorption, which in turn is affected by the Au content and TiO2 size.     

Utilization of barley straws as biosorbents for Cu2+ and Pb2+ ions

The potential to remove Cu(2+) and Pb(2+) ion from aqueous solutions through biosorption using barley straw (BS) was investigated in batch experiments. The main parameters influencing Cu(2+) and Pb(2+) ion sorption on BS were: initial metal ion concentration, amount of adsorbent, contact time and pH value of solution. The influences of initial Cu(2+) and Pb(2+) ion concentration (0.1-1mM), pH (2-9), contact time (10-240 min) and adsorbent amount (0.1-1.0 g) have been reported. Equilibrium isotherms have been measured and modelled. The percent adsorption of Cu(2+) and Pb(2+) ions increased with an increase in pH and dosage of treated BS. The biosorptive capacity of the BS was dependent on the pH of Cu(2+) and Pb(2+) ion solution. Adsorption of Cu(2+) and Pb(2+) ion was in all cases pH dependent showing a maximum at equilibrium pH value at 6.0. The equilibrium sorption capacities of Cu(2+) and Pb(2+) after 2h were 4.64 mg/g and 23.20mg/g for BS, respectively. The adsorption data fit well with the Langmuir isotherm model and the experimental result inferred that complexation on surface, adsorption (chemisorption) and ion exchange is one of the major adsorption mechanisms for binding Cu(2+) and Pb(2+) ion to the sorbents.     

Detection of heavy metal ions in water by high-resolution surface plasmon resonance spectroscopy combined with anodic stripping voltammetry

High-resolution differential surface plasmon resonance (SPR) with anodic stripping voltammetry (ASV) capability has been demonstrated for detecting heavy metal ions in water. Metal ions are electroplated onto the gold SPR sensing surface and are quantitatively detected by stripping voltammetry. Both the SPR angular shift and electrochemical current signal are recorded to identify the type and amount of the metal ions in water. The performance of the combined approach is further enhanced by a differential detection approach. The gold sensor surface is divided into a reference and a sensing area, and the difference in the SPR angles from the two areas is detected with a quadrant cell photodetector as a differential signal. Our system demonstrated quantitative detection of copper, lead, and mercury ions in water from part-per-million to sub-part-per-billion levels with good linearity.     

One pot synthesis of monodispersed L-glutathione stabilized gold nanoparticles for the detection of Pb2+ ions

Direct mixture of Au³⁺ with glutathione (GSH), which act as both reduction agents and stabilizers, in aqueous solution gave rise to production of gold nanoparticles (Au NPs) with uniform sizes of around 21 nm. The GSH stabilizer Au NPs in solution show immediate aggregation after addition of 1 mol/L NaCl aqueous solution containing Pb²⁺ ions. The Pb²⁺-induced aggregation in Au NP solution is monitored by both colorimetric response and UV-vis spectroscopy. A rather broad linear range (from 0.1 to 30 μmol/L) and low detection limit (0.1 μmol/L) are explored for Au NP sensors used for detection of Pb²⁺ ions. Furthermore, the response of GSH-stabilized Au NPs toward Pb²⁺ ions is specific compared with other possible interferants (Hg²⁺, Mg²⁺, Zn²⁺, Ni²⁺, Cu²⁺, Co²⁺, Ca²⁺, Mn²⁺, Cd²⁺, and Ba²⁺).     

Cancer biomarker detection in serum samples using surface plasmon resonance and quartz crystal microbalance sensors with nanoparticle signal amplification

Early detection of cancer is vital for the successful treatment of the disease. Hence, a rapid and sensitive diagnosis is essential before the cancer is spread out to the other body organs. Here we describe the development of a point-of-care immunosensor for the detection of the cancer biomarker (total prostate-specific antigen, tPSA) using surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) sensor platforms in human serum samples. K(D) of the antibody used toward PSA was calculated as 9.46 × 10(-10) M, indicating high affinity of the antibody used in developing the assay. By performing a sandwich assay using antibody-modified nanoparticles concentrations of 2.3 ng mL(-1) (Au, 20 nm) and 0.29 ng mL(-1) (8.5 pM) (Au, 40 nm) tPSA in 75% human serum were detected using the developed assay on an SPR sensor chip. The SPR sensor results were found to be comparable to that achieved using a QCM sensor platform, indicating that both systems can be applied for disease biomarkers screening. The clinical applicability of the developed immunoassay can therefore be successfully applied to patient's serum samples. This demonstrates the high potential of the developed sensor devices as platforms for clinical prostate cancer diagnosis and prognosis.