Picogram detection of metal ions by melanin-sensitized piezoelectric sensor

A heavy metal ion sensor was constructed by cross-linking melanin onto the gold electrode of quartz crystal microbalance (QCM). A mercury ion sensitivity of 518+/-37 Hz/ppm was observed, a substantial increase in sensitivity compared to previous reports of 10-50 Hz/ppm with the limit of detection at 5 ppb. Detection of other metal ions including Sn(2+), Ge(4+), Li(+), Zn(2+), Cu(2+), Bi(3+), Co(2+), Al(3+), Ni(2+), Ag(+), and Fe(3+) were also performed. Unexpectedly, binding of Mn(7+), Pb(2+), Cd(2+), and Cr(3+) increased resonant frequencies. The surface profile of melanin thin film upon binding to metal ions was investigated by atomic force microscopy (AFM). Structural change of melanin upon binding to metal ions was characterized by circular dichroism and by infrared spectroscopy. The current study provides the first example of melanin-coated piezoelectric sensor showing high sensitivity and selectivity to metal ions.     

Sensitivity enhancement of DNA sensors by nanogold surface modification

A novel amplified microgravimetric gene sensing system was developed using quartz crystal microbalance modified by gold nanoparticles anchored on its 1,6-hexanedithiol modified gold electrode surface, and ultrasensitive detection of DNA hybridization was accomplished at the level of at least 2 x 10(-16) M.     

Application of Polypyrrole Multi-Walled Carbon Nanotube Composite Layer for Detection of Mercury,Lead and Iron Ions Using Surface Plasmon Resonance Technique

Polypyrrole multi-walled carbon nanotube composite layers were used to modify the gold layer to measure heavy metal ions using the surface plasmon resonance technique. The new sensor was fabricated to detect trace amounts of mercury (Hg), lead (Pb), and iron (Fe) ions. In the present research, the sensitivity of a polypyrrole multi-walled carbon nanotube composite layer and a polypyrrole layer were compared. The application of polypyrrole multi-walled carbon nanotubes enhanced the sensitivity and accuracy of the sensor for detecting ions in an aqueous solution due to the binding of mercury, lead, and iron ions to the sensing layer. The Hg ion bonded to the sensing layer more strongly than did the Pb and Fe ions. The limitation of the sensor was calculated to be about 0.1 ppm, which produced an angle shift in the region of 0.3° to 0.6°.     

Modulation of base selectivity for a base-discriminating fluorescent nucleobase by addition of mercury ion

We altered the fluorescence emission selectivity of a base-discriminating fluorescent base, (Py)U, from A-selective to T-selective by the addition of mercury ion. The strong fluorescence from a duplex containing the (Py)U/T base pair was specific to the mercury ion among divalent metal ions, providing a unique method for sensing mercury ions in aqueous solutions.     

Inhibitive determination of mercury and other metal ions by potentiometric urea biosensor

The biosensor with urease entrapped in PVC layer at the surface of pH-sensitive iridium oxide electrode was applied for testing of mercury and other metal ions inhibition on enzymatic reaction. The calculation of inhibition effect was based on the measurement of initial rate of decrease of biosensor potential (proportional to the initial rate of enzymatic reaction) after addition of substrate after inhibition step. Some differences of inhibition extent were observed for various mercury forms (Hg(NO3)2, HgCl2, PhHgCl and Hg2(NO3)2) as well as for other heavy metal ions investigated as potential interferents. Because the method was not specific, it was applied for the determination of total inhibition effect caused by heavy metal ions in water samples. In the case of most cations tested the total recovery of enzyme activity was possible using Tris buffer solution with EDTA and thioacetamide after less than 10 min regeneration time.     

In vitro toxicity of palladium(II) and gold(III) porphyrins and their aqueous metal ion counterparts on Trypanosoma brucei brucei growth

The trypanocidal effects of aqueous gold(III) and palladium(II) and their metalloporphyrin derivatives on Trypanosoma brucei brucei growth in culture have been studied using an Alamar Blue indicator assay. All the experiments were conducted in the dark. As previously described for mercury(II), cadmium(II) and lead(II) porphyrins [Chem.-Biol. Interact. 139 (2002) 177], the toxicity of the metalloporphyrin complex of palladium(II) to T. b. brucei parasites was much higher compared to the aqueous free palladium(II) and free base porphyrin. Palladium(II) porphyrin, free palladium(II), and the free base porphyrin were trypanocidal to T. b. brucei at concentrations >1.5 x 10(-6), >6.1 x 10(-6) and >1.9 x 10(-5) M, respectively. While gold(III) porphyrin was effective against the parasites at concentrations >4.8 x 10(-6) M, its aqueous gold(III) was toxic at concentrations as low as 2.0 x 10(-7) M due to the generation of free radicals in the presence of this metal ion which enhanced its toxicity to the T. b. brucei parasites. Although some cell division was observed in some of the cells treated with palladium(II) porphyrin, some dividing cells had no nucleus due to unequal division and delivery of the nuclei into the daughter cells. As a result, the rate of cell division decreased with time and cell death occurred within 24 h. Interestingly, trypanosomes treated with metalloporphyrin complexes displayed different morphological features from those cells treated with free base porphyrin or metal ions. Of all the porphyrins and free metal ions tested, only mercury(II) porphyrin and aqueous gold(III) ion were toxic to the trypanosomes in the 10(-7) M range. The chemotherapeutic potential of these observations is discussed.     

Surface plasmon resonance sensing detection of mercury and lead ions based on conducting polymer composite

A new sensing area for a sensor based on surface plasmon resonance (SPR) was fabricated to detect trace amounts of mercury and lead ions. The gold surface used for SPR measurements were modified with polypyrrole-chitosan (PPy-CHI) conducting polymer composite. The polymer layer was deposited on the gold surface by electrodeposition. This optical sensor was used for monitoring toxic metal ions with and without sensitivity enhancement by chitosan in water samples. The higher amounts of resonance angle unit (ΔRU) were obtained for PPy-CHI film due to a specific binding of chitosan with Pb(2+) and Hg(2+) ions. The Pb(2+) ion bind to the polymer films most strongly, and the sensor was more sensitive to Pb(2+) compared to Hg(2+). The concentrations of ions in the parts per million range produced the changes in the SPR angle minimum in the region of 0.03 to 0.07. Data analysis was done by Matlab software using Fresnel formula for multilayer system.     

Interaction analysis of chimeric metal-binding green fluorescent protein and artificial solid-supported lipid membrane by quartz crystal microbalance and atomic force microscopy

Non-specific adsorption and specific interaction between a chimeric green fluorescent protein (GFP) carrying metal-binding region and the immobilized zinc ions on artificial solid-supported lipid membranes was investigated using the quartz crystal microbalance technique and the atomic force microscopy (AFM). Supported lipid bilayer, composed of octanethiol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyl iminodiacetic acid)succinyl] (NTA-DOGS)-Zn2+, was formed on the gold electrode of quartz resonator (5 MHz). Binding of the chimeric GFP to zinc ions resulted in a rapid decrease of resonance frequency. Reversibility of the process was demonstrated via the removal of metal ions by EDTA. Nanoscale structural orientation of the chimeric GFP on the membrane was imaged by AFM. Association constant of the specific binding to metal ions was 2- to 3-fold higher than that of the non-specific adsorption, which was caused by the fluidization effect of the metal-chelating lipid molecules as well as the steric hindrance effect. This infers a possibility for a further development of biofunctionalized membrane. However, maximization is needed in order to attain closer advancement to a membrane-based sensor device.     

An innovative spectroelectrochemical reflection cell for rapid protein electrochemistry and ultraviolet/visible/infrared spectroscopy

A novel electrochemical reflection cell combining electrochemical techniques and spectroscopy which uses a solid gold working electrode as an optical mirror is described. This cell can be used at path lengths as low as a few micrometers and thus is suitable for ultraviolet/visible (UV/Vis) and infrared spectroscopy even for aqueous solutions and suspensions. The cell was designed for small sample volumes of only a few microliters, thus reducing the effort for sample preparation. Due to the short path length of some micrometers, the entire volume is within the Nernst diffusion layer, hence resulting in fast equilibration. Evaluation of the technique is described with direct electrochemistry of horse heart cytochrome c at the gold electrode modified with 4,4'-dithiodipyridine. Cyclic voltammograms indicate rapid and reversible electrochemistry with the correct midpoint potential (52 mV vs Ag/AgCl/3 M KCl). Chronoamperometry and coulometry confirm rapid and complete oxidation and reduction; the cell volume can be entirely fully reduced within less than 10-20 s. Spectroscopy in the UV/Vis region, with potentials at the working electrode stepped between -390 and 390 mV, show perfect titration of the cytochrome c heme bands. A Nernst fit of the alpha band absorption, with redox potential Em and number of electrons n left as parameters, yields a midpoint potential of 49 mV and n=0.9. The potential of this cell in the investigation of biological electron transfer reactions and in the study of bioenergetic systems is discussed.     

Acidithiobacillus ferrooxidans fixation on mercuric surfaces and its application in stripping voltammetry

The adsorption (fixation) of bacteria Acidithiobacillus ferrooxidans on Hg and Cu metallic surfaces was qualitatively studied owing to two independent methods: frequency measurement using a quartz crystal microbalance and light absorption measuring at the Hg/bacterial culture interface. A method using a dropping mercury electrode (DME) allowed quantifying this bacterial fixation. Determining the superficial tension at Hg/bacterial culture interface led to determine bacteria adsorption on Hg through the Gibbs isotherm. A modified stripping voltammetry was proposed taking benefit of both bacterial adsorption on Hg surface and metal fixation capacity on biomass. Metal preconcentration on the biologically modified Hg electrode appeared to improve the measurement sensitivity of differential pulse anodic stripping voltammetry (DPASV). The height of the detected peaks was thus increased of 17.6% for copper, 48.4% for lead, and 132% for cadmium determinations compared to those obtained with an unmodified mercury electrode. Such augmentation depended on bulk bacteria concentration and bacteria preconcentration.     

Disruption of circulatory and respiratory activity in shore crabs (Carcinus maenas (L)) exposed to heavy metal pollution

Cardiac and respiratory activity of the shore crab, Carcinus maenas (L.), were disrupted following exposure to heavy metal ions. Exposure to 3 mg l-1 copper ions induced continuous, uninterrupted heart beat in quiescent, undisturbed crabs. Copper ions (10 mg l-1) suppressed cardiac activity and oxygen consumption within 2 hr. Alternating periods of bradycardia and tachycardia were observed together with marked changes in impedance cardiograph trace height. Similar, but more pronounced changes were seen following exposure to 1 mg l-1 mercury ions. Median perfusion index was 100 ml blood ml O2(-1) (range 58-114 ml blood ml O2(-1)) prior to pollution, but increased to peak values within the range 90-220 ml O2(-1) following exposure to copper or mercury. The effects of exposure to copper were transient and largely reversible. Exposure to mercury resulted in 100% mortality 24-48 hr after initial contamination. Death was apparently associated with loss of the osmoregulatory ability of these crabs.     

Influence of liquid medium and surface morphology on the response of QCM during immobilization and hybridization of short oligonucleotides

With the goal of developing a quartz crystal microbalance (QCM)-based DNA sensor, we have conducted an in situ QCM study along with fluorescence measurements using oligonucleotides (15-mer) as a model single-stranded DNA (ss-DNA) in two different aqueous buffer solutions; the sequence of 15-mer is a part of iduronate-2-sulphate exon whose mutation is known to cause Hunter syndrome, and the 15-mer is thiolated to be immobilized on the Au-coated quartz substrate. The fluorescence data indicate that the initial immobilization as well as the subsequent hybridization with a complementary strand is hardly dependent on the kind of buffer solution. In contrast, the mass increases deducible from the decrease of QCM frequency via the Sauerbrey equation are 2.7-6.2 and 3.0-4.4 times larger than the actual mass increases, as reflected in the fluorescence measurements, for the immobilization and the subsequent hybridization processes, respectively. Such an overestimation is attributed to the trapping of solvent as well as the formation of quite a rigid hydration layer associated with the higher viscosities and/or densities of the buffer solutions. Another noteworthy observation is the excessively large frequency change that occurs when the gold electrode is deposited in advance with Au nanoparticles. This clearly illustrates that the QCM detection of DNA hybridization is also affected greatly by the surface morphology of the electrode. These enlarged signals are altogether presumed to be advantageous when using a QCM system as an in situ probing device in DNA sensors.     

Real-time investigation of the interaction between primaquine phosphate and bovine serum albumin (BSA) by piezoelectric quartz crystal impedance analysis

Real-time investigation of the interaction between primaquine phosphate and bovine serum albumin by the piezoelectric quartz crystal impedance (PQCI) analysis was carried out for the first time. Three kinds of electrodes were investigated. Compared with bare gold (Au) electrode, the gold electrode self-assembled of nanogold colloids exhibits maintained biocompatibility, increased capacity and more bioactivity. Additionally, on the basis of the multi-dimensional information provided by the PQCI analysis, the real-time interaction information and the kinetics of the binding process was investigated and a response model was deduced. At 37 degrees C, the binding rate (k1), dissociation rate (k(-1)) and equilibrium constants (Ka) were 4.19x10(2) (mol l(-1))(-1) s(-1), 1.01x10(-3) s(-1) and 4.15x10(5) (mol l(-1))(-1) for the electrode modified by nanogold particles; 3.83x10(2) (mol l(-1))(-1) s(-1), 9.70x10(-4) s(-1) and 3.95x10(5) (mol l(-1))(-1) for the bare gold electrode, respectively.     

Application of a quartz crystal nanobalance to the molecularly imprinted recognition of phenylalanine in solution

A quartz crystal nanobalance (QCN) biosensor was developed for the selective determination of phenylalanine (Phe) in aqueous solutions. A Phe imprinted copolymer was synthesized using polyacrylonitrile and acrylic acid [poly(AN-co-AA)]. The copolymer was then coated on quartz crystal electrode to form complementary structures for the template recognition of Phe. The composite electrode was then used to determine Phe levels in solution. Determinations were based on frequency shifts of molecularly imprinted polymer (MIP) modified quartz crystal electrode caused by Phe adsorption. The frequency shifts were linearly dependent on Phe concentration over the range 50∼500 mgL⁻¹. The results obtained show that the imprinted poly(AN-co-AA) modified biosensor had higher sensitivity (0.5839 Hz/mgL⁻¹) than a non-molecularly imprinted copolymer (0.2724 Hz/mgL⁻¹). Furthermore, good reproducibility, R.S.D. = 1.84% (n = 7) was observed, and the detection limit was 45 mgL⁻¹. The selectivity of the imprinted poly(AN-co-AA) modified biosensor was examined using a number of analytes similar to Phe, i.e., dopamine (DA), ascorbic acid (AscA), vanillylmandelic acid (VMA), uric acid (UA), tryptophan (Trp), and tyrosine (Tyr), and the results obtained showed a size dependent selective effect.     

Electrochemical study of the interaction between cytochrome c and DNA at a modified gold electrode

The direct electron transfer of surface-confined horse heart cytochrome c (Cyt c) was achieved using COOH-terminated alkanethiolate-modified gold electrode. Later DNA was immobilized on the two-layer modified electrode. The quantitative determination of DNA was explored and the interaction between cytochrome c and DNA was studied. The binding site sizes were determined to be 15 bp per Cyt c molecule with double-stranded (ds) DNA and 30 nucleotides binding one Cyt c molecule with single-stranded (ss) DNA. At the dsDNA/Cyt c/MUA/Au electrode, the rate constant of oxidation electron transfer k(s,ox)=1.59x10(-3)cms-1 was obtained, at the ssDNA/Cyt c/MUA/Au electrode, the value was 2.43x10(-3)ms-1 when the scan rate was 1.0V/s. The different electrodes were characterized with electrochemical quartz crystal microbalance and atomic force microscope.     

Strategy of red blood cells immobilisation onto a gold electrode: Characterization by electrochemical impedance spectroscopy and quartz crystal microbalance

This study describes the grafting of red blood cells (RBC) onto a gold electrode. The erythrocytes were immobilised using antigen/antibody crosslinking based on the bonding of anti-D with the corresponding antigen of the RBC membrane that is shared by all erythrocytes from the positive rhesus group (O⁺). To optimise the reproducibility of the modified electrode and to avoid nonspecific interactions, the anti-D layer was deposited onto a protein G layer. The bridge between the protein G and the gold transducer was formed using mixed thiol-based self-assembled monolayer (SAM) (a mixture of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol in a 1/10 ratio). Each layer deposited was characterized, firstly, with a quartz crystal microbalance to obtain the deposited mass and the corresponding number of moles per square centimetres and secondly, by electrochemical impedance spectroscopy (EIS) using a redox couple Fe(CN)₆^3−/4− (1:1) as an EIS probe. Subsequent modelling with appropriate circuitry enabled the values for each component representing the interface (electrode/film/solution) to be calculated at each step of the grafting process. From these results the surface coverage has been calculated to range from 95 to 98%.     

Electrochemical and piezoelectric monitoring of taurine via electropolymerized molecularly imprinted films

A molecularly imprinted electrochemical quartz crystal microbalance (EQCM) sensor is fabricated here for taurine, a β ‐amino acid significant for functioning of almost all vital organs. The polymeric film of l ‐methionine was electrochemically deposited on gold‐coated EQCM electrode. Experimental parameters were optimized for controlling the performance of molecularly imprinted polymer (MIP)‐modified sensor such as ratio of monomer and template, number of electropolymerization cycles, mass deposited in each cycle, and pH. Thus, fabricated MIP‐EQCM sensor was successfully applied for estimation of taurine in solutions with varying matrices, such as aqueous, human blood plasma, milk from cow, buffalo, and milk powder. Under optimized parameters, response of MIP sensor to taurine was linearly proportional to its concentration with limit of detection as 0.12μM. Hence, a highly sensitive and selective piezoelectric sensor for taurine has been reported here via imprinting approach.     

Synthesis and unusual response to potassium of bipyridinium-benzocrown ether conjugate

A bipyridinium derivative appending a benzocrown ether, in which the phenyl unit in the benzocrown ether was directly bounded to the N-position of the bipyridinium unit, has been synthesized. The compound showed a yellow color associated with an intramolecular charge transfer (CT), which was affected by the presence of alkali and alkaline earth metal ions. An unusual CT response to K⁺ for 1 was observed and could be applicable for K⁺ sensing.     

Improvement of antibody immobilization using hyperbranched polymer and protein A

For the construction of a well-defined antibody surface, protein A was used as a binding material to immobilize antibodies onto gold-derivatized transducers. The traditional method tends to assemble protein A directly onto the gold-derivatized transducers. In this paper, we tried to indirectly bind protein A onto sensors through hyperbranched polymer (HBP) which was synthesized from p-phenylenediamine and trimesic acid. The three-dimensional structure of HBP and the characteristics including orientation control and biocompatibility of protein A led to highly efficient immunoreactions and enhanced detection system performance. With this strategy, cysteamine monolayer was first assembled onto Au electrodes associated with the piezoelectric quartz crystal; secondly, the cysteamine-modified gold electrode was further modified by the activated HBP; thirdly, protein A was immobilized onto the HBP film; and finally, antibodies were immobilized onto the surface of protein A film for detecting the corresponding antigen. The quartz crystal microbalance immunosensor thus fabricated was applied to detect hepatitis B surface antigen in solutions that ranged from 0.71 to 300 μg mL⁻¹. The detection limit was estimated to be 0.53 μg mL⁻¹. The immunosensor holds good selectivity, sensitivity, and repeatability.     

Multichannel quartz crystal microbalance array: Fabrication,evaluation, application in biomarker detection

A multichannel quartz crystal microbalance array (MQCM) with three pairs of gold electrodes was fabricated for detection of two biomarkers: acetone and nitric oxide (NO). The gold electrodes were deposited symmetrically on an AT-cut 10 MHz circular quartz plate using photolithography, sputtering, and lift-off technologies. The effect of gold layer thickness on MQCM performance was investigated and the optimized thickness was 101 nm. The simulation values of the electric parameters C₀, C_q, L_q, and R_q in the Butterworth–Van Dike equivalent circuit for the MQCM device were 97 pF, 1.3 pF, 1.05 mH, and 9.8 Ω, respectively. Simulation values were in the theoretical range, which indicated that the fabricated MQCM device had good resonance performance. Two types of nanocomposites, titanium dioxide–multiwalled carbon nanotubes and cobalt (II)phthalocyanine–silica, were synthesized as sensing materials. The sensing mechanism is based on coordination adsorption of target molecules onto the sensing material, resulting in a resonant frequency shift of modified QCM sensor. A linear range from 4.33 to 129.75 ppmv for acetone was obtained and one from 5.75 to 103.45 ppbv for NO.