Nanomolar Sensitivity Research Articles

Selective glycoprotein detection through covalent templating and allosteric click-imprinting.

Many glycoproteins are intimately linked to the onset and progression of numerous heritable or acquired diseases of humans, including cancer. Indeed the recognition of specific glycoproteins remains a significant challenge in analytical method and diagnostic development. Herein, a hierarchical bottom-up route exploiting reversible covalent interactions with boronic acids and so-called click chemistry for the fabrication of glycoprotein selective surfaces that surmount current antibody constraints is described. The self-assembled and imprinted surfaces, containing specific glycoprotein molecular recognition nanocavities, confer high binding affinities, nanomolar sensitivity, exceptional glycoprotein specificity and selectivity with as high as 30 fold selectivity for prostate specific antigen (PSA) over other glycoproteins. This synthetic, robust and highly selective recognition platform can be used in complex biological media and be recycled multiple times with no performance decrement.

Characterizing aptamer small molecule interactions with backscattering interferometry.

Aptamers are segments of single-strand DNA or RNA used in a wide array of applications, including sensors, therapeutics, and cellular process regulators. Aptamers can bind many target species, including proteins, peptides, and small molecules (SM) with high affinity and specificity. They are advantageous because they can be identified in vitro by SELEX, produced rapidly and relatively economically using oligonucleotide synthesis. The use of aptamers as SM probes has experienced a recent rebirth, and because of their unique properties they represent an attractive alternative to antibodies. Current assay methodology for characterizing small molecule-aptamer binding is limited by either mass sensitivity, as in biolayer interferometry (BLI) and surface plasmon resonance (SPR), or the need for using a fluorophore, as in thermophoresis. Here we report that backscattering interferometry (BSI), a label-free and free-solution sensing technique, can be used to effectively characterize SM-aptamer interactions, providing Kd values on microliter sample quantities and at low nanomolar sensitivity. To demonstrate this capability we measured the aptamer affinity for three previously reported small molecules; bisphenol A, tenofovir, and epirubicin showing BSI provided values consistent with those published previously. We then quantified the Kd values for aptamers to ampicillin, tetracycline and norepinephrine. All measurements produced R(2) values >0.95 and an excellent signal to noise ratio at target concentrations that enable true Kd values to be obtained. No immobilization or labeling chemistry was needed, expediting the assay which is also insensitive to the large relative mass difference between the interacting molecules.

Glutathione sulfinamide serves as a selective, endogenous biomarker for nitroxyl after exposure to therapeutic levels of donors

Nitroxyl (HNO) donors exhibit promising pharmacological characteristics for treatment of cardiovascular disorders, cancer, and alcoholism. However, whether HNO also serves as an endogenous signaling agent is currently unknown, largely because of the inability to selectively and sensitively detect HNO in a cellular environment. Although a number of methods to detect HNO have been developed recently, sensitivity and selectivity against other nitrogen oxides or biological reductants remain problematic. To improve selectivity, the electrophilic nature of HNO has been harnessed to generate modifications of thiols and phosphines that are unique to HNO, especially compared to nitric oxide (NO). Given high bioavailability, glutathione (GSH) is expected to be a major target of HNO. As a result, the putative selective product glutathione sulfinamide (GS(O)NH2) may serve as a high-yield biomarker of HNO production. In this work, the formation of GS(O)NH2 after exposure to HNO donors was investigated. Fluorescent labeling followed by separation and detection using capillary zone electrophoresis with laser-induced fluorescence allowed quantitation of GS(O)NH2 with nanomolar sensitivity, even in the presence of GSH and derivatives. Formation of GS(O)NH2 was found to occur exclusively upon exposure of GSH to HNO donors, thus confirming selectivity. GS(O)NH2 was detected in the lysate of cells treated with low-micromolar concentrations of HNO donors, verifying that this species has sufficient stability to server as a biomarker of HNO. Additionally, the concentration-dependent formation of GS(O)NH2 in cells treated with an HNO donor suggests that the concentration of GS(O)NH2 can be correlated to intracellular levels of HNO.

Electrochemical Monitoring of the Early Events of Hydrogen Peroxide Production by Mitochondria

AbstractMitochondria consume oxygen in the respiratory chain and convert redox energy into ATP. As a side process, they produce reactive oxygen species (ROS), whose physiological activities are still not understood. However, current analytical methods cannot be used to monitor mitochondrial ROS quantitatively and unambiguously. We have developed electrochemical biosensors based on peroxidase‐redox polymer‐modified electrodes, providing selective detection of H2O2 with nanomolar sensitivity, linear response over five concentration decades, and fast response time. The release of H2O2 by mitochondria was then monitored under phosphorylating or inhibited respiration conditions. We report the detection of two concomitant regimes of H2O2 release: large fluxes (hundreds of nM) under complex III inhibition, and bursts of a few nM immediately following mitochondria activation. These unprecedented bursts of H2O2 are assigned to the role of mitochondria as the hub of redox signaling in cells.

Electrochemical monitoring of the early events of hydrogen peroxide production by mitochondria.

Mitochondria consume oxygen in the respiratory chain and convert redox energy into ATP. As a side process, they produce reactive oxygen species (ROS), whose physiological activities are still not understood. However, current analytical methods cannot be used to monitor mitochondrial ROS quantitatively and unambiguously. We have developed electrochemical biosensors based on peroxidase-redox polymer-modified electrodes, providing selective detection of H2O2 with nanomolar sensitivity, linear response over five concentration decades, and fast response time. The release of H2O2 by mitochondria was then monitored under phosphorylating or inhibited respiration conditions. We report the detection of two concomitant regimes of H2O2 release: large fluxes (hundreds of nM) under complex III inhibition, and bursts of a few nM immediately following mitochondria activation. These unprecedented bursts of H2O2 are assigned to the role of mitochondria as the hub of redox signaling in cells.

Transgenic Mice for Real-Time Visualization of cGMP in Intact Adult Cardiomyocytes

3',5'-Cyclic guanosine monophosphate (cGMP) is an important second messenger that regulates cardiac contractility and protects the heart from hypertrophy. However, because of the lack of real-time imaging techniques, specific subcellular mechanisms and spatiotemporal dynamics of cGMP in adult cardiomyocytes are not well understood. Our aim was to generate and characterize a novel cGMP sensor model to measure cGMP with nanomolar sensitivity in adult cardiomyocytes. We generated transgenic mice with cardiomyocyte-specific expression of the highly sensitive cytosolic Förster resonance energy transfer-based cGMP biosensor red cGES-DE5 and performed the first Förster resonance energy transfer measurements of cGMP in intact adult mouse ventricular myocytes. We found very low (≈10 nmol/L) basal cytosolic cGMP levels, which can be markedly increased by natriuretic peptides (C-type natriuretic peptide >> atrial natriuretic peptide) and, to a much smaller extent, by the direct stimulation of soluble guanylyl cyclase. Constitutive activity of this cyclase contributes to basal cGMP production, which is balanced by the activity of clinically established phosphodiesterase (PDE) families. The PDE3 inhibitor, cilostamide, showed especially strong cGMP responses. In a mild model of cardiac hypertrophy after transverse aortic constriction, PDE3 effects were not affected, whereas the contribution of PDE5 was increased. In addition, after natriuretic peptide stimulation, PDE3 was also involved in cGMP/cAMP crosstalk. The new sensor model allows visualization of real-time cGMP dynamics and pharmacology in intact adult cardiomyocytes. Förster resonance energy transfer imaging suggests the importance of well-established and potentially novel PDE-dependent mechanisms that regulate cGMP under physiological and pathophysiological conditions.

A Highly Selective Biosensor with Nanomolar Sensitivity Based on Cytokinin Dehydrogenase

We have developed a N 6-dimethylallyladenine (cytokinin) dehydrogenase-based microbiosensor for real-time determination of the family of hormones known as cytokinins. Cytokinin dehydrogenase from Zea mays (ZmCKX1) was immobilised concurrently with electrodeposition of a silica gel film on the surface of a Pt microelectrode, which was further functionalized by free electron mediator 2,6-dichlorophenolindophenol (DCPIP) in supporting electrolyte to give a bioactive film capable of selective oxidative cleavage of the N 6- side chain of cytokinins. The rapid electron shuffling between freely diffusible DCPIP and the FAD redox group in ZmCKX1 endowed the microbiosensor with a fast response time of less than 10 s. The immobilised ZmCKX1 retained a high affinity for its preferred substrate N 6-(Δ2-isopentenyl) adenine (iP), and gave the miniaturized biosensor a large linear dynamic range from 10 nM to 10 µM, a detection limit of 3.9 nM and a high sensitivity to iP of 603.3 µAmM−1cm−2 (n = 4, R2 = 0.9999). Excellent selectivity was displayed for several other aliphatic cytokinins and their ribosides, including N 6-(Δ2-isopentenyl) adenine, N 6-(Δ2-isopentenyl) adenosine, cis-zeatin, trans-zeatin and trans-zeatin riboside. Aromatic cytokinins and metabolites such as cytokinin glucosides were generally poor substrates. The microbiosensors exhibited excellent stability in terms of pH and long-term storage and have been used successfully to determine low nanomolar cytokinin concentrations in tomato xylem sap exudates.

Nanomolar Pyrophosphate Detection in Water and in a Self-Assembled Hydrogel of a Simple Terpyridine-Zn2+ Complex

A simple terpyridine-Zn(II) complex is shown to act as an efficient and highly selective fluorescent sensor for pyrophosphate in water at physiological pH. The sensor complex showed an unprecedented fluorescence response (∼500 fold increase) and a record nanomolar sensitivity (detectable fluorescent response at 20 nM and LOD ∼ 0.8 nM). It has successfully been used to stain and record confocal fluorescence microscopy images of HeLa cells. Moreover, the complex was found to self-assemble into a hydrogel which was subsequently used to coat disposable paper strips for easy, low-cost detection of pyrophosphate.

Detection of miRNA Expression in Intact Cells Using Activatable Sensor Oligonucleotides

We describe a technology for the profiling of miRNA expression in intact cells. The technology is based on sensor oligonucleotides that are cleavable, completely complementary to a target miRNA, and dual-labeled with a fluorescent dye and a quencher. Upon entering the cell, the sensor oligonucleotide binds its specific miRNA target through complementary base-pairing. This triggers assembly of the endogenous RNA Induced Silencing Complex (RISC) around the miRNA-sensor duplex and cleavage of the sensor oligonucleotide, resulting in separation between the dye and quencher, and a fluorescence turn-on. In the presented feasibility studies, we focus on a specific miRNA (miR-10b) implicated in breast cancer metastasis. Using a human breast adenocarcinoma cell line, we illustrate the application of this technology for miRNA detection with nanomolar sensitivity in both a cell-free system and intact cells.

A remarkable effect of N,N-diethylamino functionality on the optoelectronic properties of a salicylimine-based probe for Al3+

A new live-cell permeable, fluorescent probe comprised of a simple salicylimine-based Schiff base (SA1) has been developed for Al3+ with nano-molar sensitivity in aqueous media. SA1 was synthesized through a simple structural modification of a recently reported receptor SA2 by the incorporation of the N,Ndiethylamino (DEA) group as a fine controllable unit. This modification affects the performance of SA1 remarkably in terms of its sensitivity, water compatibility and efficiency as well as its mechanistic aspect. The presence of the DEA group in SA1 led to its dual channel emission due to the TICT state and at the same time its hydrophobic nature was also responsible for controlling the strong hydration of Al3+ ions in aqueous media which ultimately led to the high sensitivity of SA1 for Al3+. The structure of SA1 was confirmed by single crystal X-ray diffraction and its binding with Al3+ was studied in detail using UVvisible, fluorescence and 1H NMR spectral studies along with mass determination. The effort of getting a single crystal of Al3+–SA1 led to single crystals of Cl−/NO3 − complexes of protonated SA1 which were fully characterized by their XRD studies.

English

Photoacoustic imaging, also called optoacoustic imaging, is a new biomedical imaging modality based on the use of laser-generated ultrasound. It is a hybrid modality, combining the high- contrast and spectroscopy based specificity of optical imaging with the high spatial resolution of ultrasound imaging. In essence, a Photoacoustic image can be regarded as an ultrasound image in which the contrast depends not on the mechanical and elastic properties of the tissue, but its optical properties, specifically optical absorption. As a result, it offers greater specificity than conventional sonographic imaging with the ability to detect haemoglobin, lipids, water and other light-absorbing chromophores, but with greater penetration depth than purely optical imaging modalities that rely on ballistic photons. In addition to visualizing anatomical structures such as the microvasculature, it can also provide functional information such as blood oxygenation, blood flow and temperature. These attributes make photoacoustic imaging applicable in clinical medicine, preclinical research and early detection of cancer, cardiovascular disease and abnormalities of microcirculation. Photoacoustic microscopy is a promising tool for imaging both dental decay and dental pulp. Using photoacoustics, near-infrared optical contrast between sound and carious dental tissues can be detected relatively easily and accurately at ultrasound resolution and may ultimately allow for continuous monitoring of caries before and during treatment. Photoacousting imaging compares favorably to other imaging modalities with its precise depth information, submillimeter resolution, and nanomolar sensitivity. With further improvement in background reduction, as well as the use of lasers with high-repetition rates, it is likely that Photoacoustic imaging will find wide use in the future in both basic research and clinical care. It is a highly vibrant research field in the years to come. This paper intends to discuss recent technical progress in photoacousting imaging and presents corresponding applications. INTRODUCTION: Photoacoustic imaging, an emerging hybrid imaging modality that can provide strong endogenous and exogenous optical absorption contrasts with high ultrasonic spatial resolution using the photoacoustic (PA) effect, has overcome the fundamental depth limitation. The image resolution is scalable with the ultrasonic frequency. The imaging depth is limited to the reach of photons and up to a few centimeters deep in biological tissues.Possessing many attractive characteristics such as the use of nonionizing electromagnetic waves, good resolution and contrast, portable instrumentation, and the ability to partially quantitate the signal, photoacoustic techniques have been applied to the imaging of cancer, wound healing, disorders in the brain, and gene expression, among others. As a promising structural, functional, and molecular imaging modality for a wide range of biomedical applications, photoacoustic imaging can be categorized into two types of systems: photoacoustic computed tomography (PACT) and photoacoustic microscopy (PAM).The photoacoustic computed tomography (PACT), which uses reconstruction algorithms to generate an

High-Sensitivity Monoclonal Antibodies Specific for Homoserine Lactones Protect Mice from Lethal Pseudomonas aeruginosa Infections

A number of bacteria, including pathogens like Pseudomonas aeruginosa, utilize homoserine lactones (HSLs) as quorum sensing (QS) signaling compounds and engage in cell-to-cell communication to coordinate their behavior. Blocking this bacterial communication may be an attractive strategy for infection control as QS takes a central role in P. aeruginosa biology. In this study, immunomodulation of HSL molecules by monoclonal antibodies (MAbs) was used as a novel approach to prevent P. aeruginosa infections and as tools to detect HSLs in bodily fluids as a possible first clue to an undiagnosed Gram-negative infection. Using sheep immunization and recombinant antibody technology, a panel of sheep-mouse chimeric MAbs were generated which recognized HSL compounds with high sensitivity (nanomolar range) and cross-reactivity. These MAbs retained their nanomolar sensitivity in complex matrices and were able to recognize HSLs in P. aeruginosa cultures grown in the presence of urine. In a nematode slow-killing assay, HSL MAbs significantly increased the survival of worms fed on the antibiotic-resistant strain PA058. The therapeutic benefit of these MAbs was further studied using a mouse model of Pseudomonas infection in which groups of mice treated with HSL-2 and HSL-4 MAbs survived, 7 days after pathogen challenge, in significantly greater numbers (83 and 67%, respectively) compared with the control groups. This body of work has provided early proof-of-concept data to demonstrate the potential of HSL-specific, monoclonal antibodies as theranostic clinical leads suitable for the diagnosis, prevention, and treatment of life-threatening bacterial infections.

9, 10-Bis(8-Quinolinoxymethyl)Anthracene—A Fluorescent Sensor for Nanomolar Detection of Cu2+ with Unusual Acid Stability of Cu2+-Complex

In this work, the dipod 9,10-bis(8-quinolinoxymethyl)anthracene (1) and for comparison, monopod 9-(8-quinolinoxymethyl)anthracene (2) have been synthesized. The fluoroionophore 1 in pH 7.1 HEPES buffered CH3CN:H2O (4:1 v/v) solution shows quenching only with Cu(2+) with lowest limit of detection 150 nM, amongst various metal ions. Fluoroionophore 1 could also be applied to sense Co(2+) with lowest limit of detection 600 nM. By modulating the pH of the solution and concentration of Cu(2+), 1 shows respective "On-Off-On" and "On-Off" fluorescent switching. The self-assembly of two Cu(2+) ions and two molecules of fluoroionophore 1 to form closed structure [Cu2(L)2](4+) seems to be responsible for nanomolar sensitivity towards Cu(2+). The combination of delayed second protonation of 1 (pK(a2) = 2.6) and stepwise protonation of [Cu2 L2](4+) causes unusual stability of [Cu(LH)2](4+) even at pH < 2.

Photoelectrochemical oxidation of a turn-on fluorescent probe mediated by a surface MnII catalyst covalently attached to TiO2 nanoparticles

A manganese complex covalently attached to a TiO2 electrode via a light-absorbing organic linker (L) was used in the photooxidation of 2′,7′-dihydrodichlorofluorescein (H2DCF). Significant and sustained photocurrent was observed upon visible-light illumination of the fully assembled anode in the presence of the substrate. The two-electron, two-proton oxidation of H2DCF yields the fluorescent compound, 2′,7′-dichlorofluorescein (DCF). Our studies suggest that the MnII–L–TiO2 architecture is an effective photoanode for multielectron chemistry, as production of DCF under visible-light illumination exceeds yields observed for bare TiO2 as well as ZnII–L–TiO2 anodes. The turn-on fluorescent behavior of H2DCF upon oxidation makes it an excellent substrate for the study of new photoanodes. The high fluorescence quantum yield of DCF allows for nanomolar sensitivity and real-time monitoring of substrate oxidation.

Multiplexed Plasmon Sensor for Rapid Label-Free Analyte Detection

Efficient and cost-effective multiplexed detection schemes for proteins in small liquid samples would bring drastic advances to fields like disease detection or water quality monitoring. We present a novel multiplexed sensor with randomly deposited aptamer functionalized gold nanorods. The spectral position of plasmon resonances of individual nanorods, monitored by dark-field spectroscopy, respond specifically to different proteins. We demonstrate nanomolar sensitivity, sensor recycling, and the potential to upscale to hundreds or thousands of targets.

Identification of a critical region in the Drosophila ryanodine receptor that confers sensitivity to diamide insecticides

Anthranilic diamides, which include the new commercial insecticide, chlorantraniliprole, are an exciting new class of chemistry that target insect ryanodine receptors. These receptors regulate release of stored intracellular calcium and play a critical role in muscle contraction. As with insects, nematodes express ryanodine receptors and are sensitive to the plant alkaloid, ryanodine. However the plant parasitic nematode, Meloidogyne incognita, is insensitive to anthranilic diamides. Expression of a full-length Drosophila melanogaster ryanodine receptor in an insect cell line confers sensitivity to the receptor agents, caffeine and ryanodine along with nanomolar sensitivity to anthranilic diamides. Replacement of a 46 amino acid segment in a highly divergent region of the Drosophila C-terminus with that from Meloidogyne results in a functional RyR which lack sensitivity to diamide insecticides. These findings indicate that this region is critical to diamide sensitivity in insect ryanodine receptors. Furthermore, this region may contribute to our understanding of the differential selectivity diamides exhibit for insect over mammalian ryanodine receptors.

Size‐dependent ferrohydrodynamic relaxometry of magnetic particle imaging tracers in different environments

Magnetic particle imaging (MPI) is a recently developed imaging technique that seeks to provide ultrahigh resolution and tracer sensitivity with positive contrast directly originated from superparamagnetic iron oxide nanoparticles (NPs). MPI signals can be generated from a combination of Néel relaxation, Brownian rotational diffusion, and hysteretic reversal mechanisms of NPs in response to applied magnetic fields. When specific targeting of organs, such as carcinoma and endothelial cardiovascular cells, is needed, different behavior may be expected in immobilized NPs, due to complete or partial elimination of the Brownian motion. Here, the authors present an experimental investigation of the MPI spatial resolution and signal intensities as a function of a wide range of median core sizes of NPs under four representative conditions, including after immobilization in a tissue equivalent medium. Monodisperse hydrophobic NPs with median core diameters (d0) ranging from 7 to 22 nm were synthesized in organic media and subsequently dispersed in aqueous solution after a facile surface modification. Morphology, median size, size distribution, and magnetic properties of the NPs were investigated. Hydrophobic and hydrophilic NPs with various core sizes were immobilized in trioctyl phosphine oxide and agarose gel, respectively. Their size-dependent performance as MPI tracers for system matrix and x-space image reconstruction was evaluated using magnetic particle spectrometry (MPS) and compared with the free rotating counterparts. Immobilized NPs with core diameters smaller than ≈ 20 nm have similar spatial resolution, but lower signal intensities when compared with their free rotating counterparts. Compared to their performance in solution, spatial resolution was improved, but signal intensity was lower, when larger NPs with core size of 22 nm were immobilized in agarose. Same trends were observed in signal intensities, when considering either system matrix or x-space approaches. The harmonic and dm/dH signal intensities changed linearly and the spatial resolution did not change with decreasing NP concentration up to 15 μg/ml. The results show that the MPI signal is very sensitive to both NP size and environment. The authors' calculations show that Brownian rotational diffusion is slower than the field switching cycle and, therefore, it has minimal influence on MPS signals. dm/dH analyses show that Néel relaxation is the dominant mechanism determining MPI response in smaller NPs (d0 < ≈ 20 nm). Larger NPs show hysteretic reversal when the applied field amplitude is large enough to overcome the coercivity. Linear variation of the MPS signal intensity with iron concentration but with uniform spatial resolution enables quantitative imaging for a range of applications, from high-concentration bolus chase imaging to low-concentration molecular imaging (while the authors' instrument is noise-limited to ≈ millimolar iron concentrations, nanomolar sensitivity is expected for MPI, theoretically). These results pave the way for future application of the authors' synthesized tracers for immobilized or in vivo targeted MPI of tissues.

Inhibitor profile of bis(n)-tacrines and N-methylcarbamates on acetylcholinesterase from Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi

The cattle tick, Rhipicephalus (Boophilus) microplus (Bm), and the sand fly, Phlebotomus papatasi (Pp), are disease vectors to cattle and humans, respectively. The purpose of this study was to characterize the inhibitor profile of acetylcholinesterases from Bm (BmAChE1) and Pp (PpAChE) compared to human and bovine AChE, in order to identify divergent pharmacology that might lead to selective inhibitors. Results indicate that BmAChE has low sensitivity (IC50=200μM) toward tacrine, a monovalent catalytic site inhibitor with sub micromolar blocking potency in all previous species tested. Similarly, a series of bis(n)-tacrine dimer series, bivalent inhibitors and peripheral site AChE inhibitors possess poor potency toward BmAChE. Molecular homology models suggest the rBmAChE enzyme possesses a W384F orthologous substitution near the catalytic site, where the larger tryptophan side chain obstructs the access of larger ligands to the active site, but functional analysis of this mutation suggests it only partially explains the low sensitivity to tacrine. In addition, BmAChE1 and PpAChE have low nanomolar sensitivity to some experimental carbamate anticholinesterases originally designed for control of the malaria mosquito, Anopheles gambiae. One experimental compound, 2-((2-ethylbutyl)thio)phenyl methylcarbamate, possesses >300-fold selectivity for BmAChE1 and PpAChE over human AChE, and a mouse oral LD50 of >1500mg/kg, thus providing an excellent new lead for vector control.

Determination of ferric ions using surface-enhanced Raman scattering based on desferrioxamine-functionalized silver nanoparticles

A simple, robust and reproducible spectroscopic method based on surface-enhanced Raman scattering with nanomolar sensitivity has been developed for selective iron(III) determination in aqueous solutions.

A novel calix[4]arene thiol functionalized silver nanoprobe for selective recognition of ferric ion with nanomolar sensitivity via DLS selectivity in human biological fluid

A high concern for human health and safety has motivated dynamic research on the potential impact of transition metal ions and their toxic effects, thus it is very challenging to design transition-metal ion detection devices that are cost-effective, rapid and applicable to the biological milieus. Driven by the need to detect trace amounts of Fe(3+) from blood samples, we report a highly selective and ultrasensitive calix[4]arene modified silver nanoprobe for Fe(3+) recognition at the 9.4 nM level from aqueous solution with excellent discrimination against other heavy metals and biomolecules. The assembly was characterized by TEM (transmission electron microscopy), DLS (dynamic light scattering), UV-Vis, FT-IR, ESI-MS and (1)H NMR spectrometry, which demonstrate the higher binding affinity for Fe(3+). The biosensor has been successfully applied to estimate the ferric ion in human blood serum as well as in human hemoglobin.