Biosensor recognition of thyroid-disrupting chemicals using transport proteins

Novel surface plasmon resonance-based biosensor assays for the bioeffect-related screening of chemicals with thyroid-disrupting activity are described. Two thyroid transport proteins (TPs), thyroxine binding globulin (TBG) and recombinant transthyretin (rTTR), were applied in an inhibition assay format in a Biacore 3000 using CM5 biosensor chips coated with l-thyroxine (T4), the main hormone of the thyroid system. Assay conditions were optimized for the natural thyroid hormones, and known thyroid disruptors and structurally related compounds were selected as model compounds to be tested in both assays for their relative potency (RP) compared to T4. The chosen compounds were halogenated phenols, halogenated bisphenols, bisphenol A, 3,5-dichlorobiphenyl, and its hydroxylated metabolite 4-hydroxy-3,5-dichlorobiphenyl (4-OH PCB 14). The TBG-based assay was highly specific for T4, and the rTTR-based assay was sensitive toward several compounds, the highest sensitivity (RP = 4.4) being obtained with 4-OH PCB 14, followed by tetrabromobisphenol A (RP = 1.5) and tetrachlorobisphenol A (RP = 0.75). For the bioeffect-related screening of known and identification of possible new thyroid disruptors, the TPs-based biosensor assays were more sensitive (IC(50) of 13.7 +/- 1.3 and 8.6 +/- 0.7 nM for the rTTR and the TBG-based assay, respectively), easier to perform, and faster alternatives (10 min/sample) than the currently used methods such as radioligand binding assays and immunoprecipitation-HPLC.     

Drug of abuse confirmation in human urine using stepwise solid-phase extraction and micellar electrokinetic capillary chromatography.

This paper demonstrates that most common drugs of abuse can be absorbed simultaneously onto a mixed-mode bonded-phase matrix and eluted sequentially in two to three steps for subsequent analysis by micellar electrokinetic capillary chromatography (MECC). Having on-column multiwavelength UV absorption detection, this is shown to be an attractive approach for confirmation testing of barbiturates, hypnotics, amphetamines, opioids, benzodiazepines, and metabolites of cocaine in a single aliquot of human urine. For these compounds, no hydroysis of the urine specimen or sample derivatization is required. Under the examined conditions using 5 mL of urine, excellent recoveries (80-90% level) and detection limits (about 100 ng/mL) are obtained. For patient urines which tested positively for different classes of drugs using immunological screening methods, a two-step extraction scheme is shown to provide extracts suitable for rapid MECC confirmation of the drugs of abuse.     

Bioassay-directed identification of estrogen residues in urine by liquid chromatography electrospray quadrupole time-of-flight mass spectrometry

A new approach to the search for residues of known and unknown estrogens in calf urine is presented. Following enzymatic deconjugation and solid-phase extraction, a minor part of the samples is screened for estrogen activity using a recently developed rapid reporter gene bioassay. The remainder of the bioactive extracts is analyzed by gradient liquid chromatography (LC) with, in parallel, bioactivity and mass spectrometric detection via effluent splitting toward a 96-well fraction collector and an electrospray quadrupole time-of-flight mass spectrometer (QTOFMS). The LC fractions in the 96-well plate are used for the detection of estrogen activity using the bioassay. The biogram obtained features a 20-s time resolution, and the suspect well numbers can be easily correlated with the LC/QTOFMS retention time. The mass spectral data from the thus assigned relevant parts of the chromatograms are background subtracted, followed by accurate mass measurement, element composition calculation, and identification. The method allows estrogen activity detection and identification of (un)known estrogens in urine at the 1-2 ng/L level, in compliance with current residue analysis performance for hormone abuse in cattle. The applicability of this LC/bioassay/QTOFMS approach for the identification of estrogens in real-life samples is demonstrated by the analysis of several calf urine samples, and preliminary data from a pig feed sample.     

Urine testing for designer steroids by liquid chromatography with androgen bioassay detection and electrospray quadrupole time-of-flight mass spectrometry identification

New anabolic steroids show up occasionally in sports doping and in veterinary control. The discovery of these designer steroids is facilitated by findings of illicit preparations, thus allowing bioactivity testing, structure elucidation using NMR and mass spectrometry, and final incorporation in urine testing. However, as long as these preparations remain undiscovered, new designer steroids are not screened for in routine sports doping or veterinary control urine tests since the established GC/MS and LC/MS/MS methods are set up for the monitoring of a few selected ions or MS/MS transitions of known substances only. In this study, the feasibility of androgen bioactivity testing and mass spectrometric identification is being investigated for trace analysis of designer steroids in urine. Following enzymatic deconjugation and a generic solid-phase extraction, the samples are analyzed by gradient LC with effluent splitting toward two identical 96-well fraction collectors. One well plate is used for androgen bioactivity detection using a novel robust yeast reporter gene bioassay yielding a biogram featuring a 20-s time resolution. The bioactive wells direct the identification efforts to the corresponding well numbers in the duplicate plate. These are subjected to high-resolution LC using a short column packed with 1.7-microm C18 material and coupled with electrospray quadrupole time-of-flight mass spectrometry (LC/QTOFMS) with accurate mass measurement. Element compositions are calculated and used to interrogate electronic substance databases. The feasibility of this approach for doping control is demonstrated via the screening of human urine samples spiked with the designer anabolic steroid tetrahydrogestrinone. Application of the proposed methodology, complementary to the established targeted urine screening for known anabolics, will increase the chance of finding unknown emerging designer steroids, rather then being solely dependent on findings of the illicit preparations themselves.     

Nanoscale affinity chip interface for coupling inhibition SPR immunosensor screening with Nano-LC TOF MS

The on-line nanoscale coupling of a surface plasmon resonance (SPR)-based inhibition biosensor immunoassay (iBIA) for the screening of low molecular weight molecules with nano-liquid-chromatography electrospray ionization time-of-flight mass spectrometry (nano-LC ESI TOF MS) for identification is described. The interface is based on a reusable recovery chip (RC) that contains a nanoscale biosorbent composed of a hydrogel layer modified with antibodies raised against the analyte featuring the unique possibility of performance characterization using the SPR biosensor. Various hydrogel chemistries were evaluated, and the standard Biacore CM5 chip showed the highest capture capacity in combination with affinity-purified polyclonal antibodies. The procedure has four stages: the samples are prepared (1) and screened using a screening chip (SC) in the iBIA (2). Suspected noncompliant samples as being noncompliant are reinjected over the RC, and the analyte is captured at subnanogram level (3). The captured analyte is released, and the eluate is analyzed with nano-LC ESI TOF MS via a loop-type interface (4). The coupling of the technologies proved effective for screening enrofloxacin, a model compound, in incurred chicken muscle samples followed by identity confirmation in suspected noncompliant samples. Ciprofloxacin, a known metabolite of enrofloxacin, was identified as well in incurred chicken samples. This demonstrates the potential of the technologies coupled by means of a RC for the rapid screening and identification of known as well as unknown compounds. Finally, we demonstrate the feasibility of combining the two biosensor chips (SC and RC) with a robust chip-based nano-LC chip TOF MS system, thus providing a robust alternative triple-chip system.     

Rapid screening of anabolic steroids in urine by reactive desorption electrospray ionization

Fast screening for anabolic steroids in whole urine is achieved by combining reactive desorption electrospray ionization (reactive DESI) and tandem mass spectrometry. Spray solutions containing hydroxylamine allow heterogeneous reactions of hydroxylamine with the carbonyl group of the steroids during the ionization process. Seven steroids, including a glycosteroid, were examined. The ion/molecule reaction adduct and the oxime formed via its dehydration were observed using reactive DESI; the protonated and sodiated forms of the ionized steroid were also observed both in reactive DESI and in DESI performed without the added hydroxylamine reagent. Paper, glass, and polytetrafluoroethylene were tested as sample substrates, but the glycosteroid was ionized intact without hydrolysis only from polytetrafluoroethylene. Limits of detection for the pure compounds were less than 1 ng, dynamic ranges were typically 2 orders of magnitude, and analysis times were just a few seconds. Concentration levels of ketosteroids in raw urine relevant to screening for sports doping (approximately 20 ng/mL) can be reached using a simple solid-phase microextraction (SPME) preconcentration step. Reactive DESI provided significant improvements in ionization efficiency of these steroids in raw undiluted urine as compared to conventional DESI; suppression effects due to the sample matrix were minimal and the urine matrix had no deleterious effect on steroid detection limits. Tandem mass spectrometry provided confirmation of analyte identification in this rapid screening process.     

A kinetic study of analyte-receptor binding and dissociation for surface plasmon resonance biosensors applications

A fractal analysis, which takes into account the effect of surface heterogeneity brought about by ligand immobilization on the reaction kinetics in surface plasmon resonance (SPR) biosensors, is presented. The binding and dissociation of estrogen receptors (ERs), ERa and ER/spl alpha/ and ER/spl beta/, in solution to different ligands immobilized on the SPR biosensor is analyzed within the fractal framework. The heterogeneity on the biosensor surface is made quantitative by using a single number, the fractal dimension D/sub f/. The analysis provides physical insights into the binding of these receptors to different ligands and compounds, particularly the endocrine disrupting compounds (EDCs). These EDCs have deleterious effects on humans and on wildlife. Single- and dual-fractal models were employed to fit the ER-binding data obtained from the literature. Values of the binding and dissociation rate coefficient and fractal dimensions were obtained from a regression analysis provided by Corel Quattro Pro, 8.0. Values for the affinity K/sub D/(=k/sub d//k/sub a/) were also calculated. This provides us with some extra flexibility in designing biomolecular assays. The analysis should provide further information on the mode of action and interaction of EDCs with the ERs. This would help in the design of agents and modulators against these EDCs.     

Simultaneous use of time-resolved fluorescence and anti-stokes photoluminescence in a bioaffinity assay

A bioaffinity assay is described where anti-Stokes photoluminescence of inorganic lanthanide phosphors and time-resolved fluorescence of lanthanide chelates are measured from a single microtitration well without any disturbance from these label technologies to each other. Up-converting phosphor (UPC-phosphor) bioconjugate was produced by grinding the commercial, micrometer-sized UPC-phosphors to colloidal, submicrometer-sized phosphor particles and by attaching these phosphors to biomolecules. Experiments were carried out in standard 96-well microtitration plates to determine detection limits, linearity, and cross-talk of UPC-phosphor and europium chelate. In numbers of molecules the lower limits of detection for UPC-phosphor were roughly 3 x 10(3) particles in solution and 1 x 10(4) particles in solid phase, and for europium label same values were 9 x 10(6) and 9 x 10(7) molecules. Linearity of detection was for UPC-phosphor 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase and for europium label over 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase. The cross-talk between the two labels was practically nonexistent. In this study we show that up-converting anti-Stokes photoluminescent phosphors could be employed in bioaffinity assays as very potential labels with significant advantages either alone or together with long-lifetime lanthanide chelates.     

Rapid screening of doping agents in human urine by vacuum MALDI-linear ion trap mass spectrometry

Detection of doping agents in urine frequently requires extensive separation prior to chemical analyses. Gas or liquid chromatography coupled to mass spectrometry has produced accurate and sensitive assays, but chromatographic separations require time and, sometimes, chemical derivatization. To avoid such tedious and lengthy procedures, vacuum matrix-assisted laser desorption ionization (vMALDI) coupled with the linear ion trap mass spectrometry (LIT/MS) technique is tested for its applicability as a rapid screening technique. Commonly used doping agents like nandrolone, boldenone, trenbolone, testosterone, and betamethasone were chosen as study compounds. Different MALDI matrixes like alpha-cyano-4-hydroxycinnamic acid (CHCA), dihyroxy benzoic acid (DHB) with and without cetyl trimethyl ammonium bromide (CTAB), a surfactant, and meso-tetrakis(pentafluorophenyl) porphyrin (F20TPP) were tested. Among them, F20TPP (MW 974.57 Da) was selected as the preferred matrix owing to the lack of interfering matrix peaks at the lower mass range (m/z 100-700). Urine samples spiked with study compounds were processed by solid-phase extraction (SPE) and consistently detected through a linear range of 0.1-100 ng/mL. The limit of detection and lower limit of quantification for all five analytes have been determined to be 0.03 and 0.1 ng/mL, respectively, in urine samples. Testosterone-d3 was used as an internal standard, and the quantitative measurements were achieved by the selective reaction monitoring (SRM) mode. The method was validated and showed consistency in the results. Hence, vMALDI-LIT/MS can be used as a rapid screening method to complement the traditional GC/MS and LC/MS techniques for simultaneous identification, confirmation, and quantification of doping agents in urine.     

Protein-doped monolithic silica columns for capillary liquid chromatography prepared by the sol-gel method: applications to frontal affinity chromatography

The development of bioaffinity chromatography columns that are based on the entrapment of biomolecules within the pores of sol-gel-derived monolithic silica is reported. Monolithic nanoflow columns are formed by mixing the protein-compatible silica precursor diglycerylsilane with a buffered aqueous solution containing poly(ethylene oxide) (PEO, MW 10,000) and the protein of interest and then loading this mixture into a fused-silica capillary (150-250-microm i.d.). Spinodal decomposition of the PEO-doped sol into two distinct phases prior to the gelation of the silica results in a bimodal pore distribution that produces large macropores (>0.1 microm), to allow good flow of eluent with minimal back pressure, and mesopores (approximately 3-5-nm diameter) that retain a significant fraction of the entrapped protein. Addition of low levels of (3-aminopropyl)triethoxysilane is shown to minimize nonselective interactions of analytes with the column material, resulting in a column that is able to retain small molecules by virtue of their interaction with the entrapped biomolecules. Such columns are shown to be suitable for pressure-driven liquid chromatography and can be operated at relatively high flow rates (up to 500 microL x min(-1)) or with low back pressures (<100 psi) when used at flow rates of 5-10 microL x min(-1). The clinically relevant enzyme dihydrofolate reductase was entrapped within the bioaffinity columns and was used to screen mixtures of small molecules using frontal affinity chromatography with mass spectrometric detection. Inhibitors present in compound mixtures were retained via bioaffinity interactions, with the retention time being dependent on both the ligand concentration and the affinity of the ligand for the protein. The results suggest that such columns may find use in high-throughput screening of compound mixtures.     

Biologically produced bifunctional recombinant protein nanoparticles for immunoassays

Nanoparticles are increasingly used as labels for analytical purposes. In general, nanoparticles need to be functionalized with binding molecules (mostly antibodies or fragments thereof) and label substances using a multistep process that requires several manufacturing and purification steps. Here, we present a biological method of producing functionalized nanoparticles for effective use as label agents in a bioaffinity assay. The particles are based on the globular protein shell of human ferritin. A single chain Fv fragment (scFv) of an antibody is used as the binding moiety and Eu3+ ions as the label substance. Conventional chemical conjugation of the particle and antibody fragment is replaced with genetic fusion between the ferritin subunit and scFv genes. The material, for example, the fusion construct is produced in a single bacterial culture as insoluble forms that are easily purified by centrifugations. The subunits are solubilized and self-assembled, and label ions are introduced by shifting the pH. The functionality of these particles is demonstrated with a bioaffinity assay. This method of producing nanoparticles with inherent antigen binding activity presents several possibilities for the simple production of specific, functional nanoparticles. Production is fast, economical, and environmentally sustainable, making the system advantageous, particularly in applications requiring large quantities of specific nanoparticles.     

Potentiality of gas chromatography-triple quadrupole mass spectrometry in vanguard and rearguard methods of pesticide residues in vegetables

A new analytical strategy for the screening and confirmation/quantification of multiclass pesticide residues in vegetables has been established and validated. No complicated sample preparation was needed, but only a simple and rapid extraction using ethyl acetate and sodium sulfate, which required no cleanup. The approach is based on the use of the triple quadrupole (QqQ) mass spectrometry (MS) as detection system in gas chromatography (GC). In a first step, a GC-QqQ-MS screening method, which monitors only one MS/MS transition by compound, allows the identification of approximately 130 pesticides in 11.6 min. In this way, the differentiation between negative and potentially nonnegative samples is carried out. In the second step, the nonnegative samples are reanalyzed by the GC-QqQ-MS confirmation/quantification method, which monitors two or three MS/MS transitions by compound. Confirmation of pesticides was based on the comparison of intensity ratios for the main ions in samples with those obtained on the same day from the standard in a matrix containing the pesticides at a preestablished concentration level. Quantification of the identified and confirmed pesticides was based on the addition standard method, which avoids matrix effect. The proposed analytical strategy allowed a reliable identification and confirmation of the target pesticides at trace levels, reducing analysis time and increasing sample throughput in routine analytical laboratories.     

The methods of identification,analysis, and removal of endocrine disrupting compounds (EDCs) in water

The information regarding endocrine disrupting compounds (EDCs) was reviewed, including the definition and characteristics, the recent research trends concerning identification and analytical methods, and the applicable removal processes. EDCs include various types of natural and synthetic chemical compounds presenting the mimicking or inhibition of the reproductive action of the endocrine system in animals and humans. The ubiquitous presence with trace level concentrations and the wide diversity are the reported characteristics of EDCs. Biologically based assays seem to be a promising method for the identification of EDCs. On the other hand, mass-based analytical methods show excellent sensitivity and precision for their quantification. Several extraction techniques for the instrumental analysis have been developed since they are crucial in determining overall analytical performances. Conventional treatment techniques, including coagulation, precipitation, and activated sludge processes, may not be highly effective in removing EDCs, while the advanced treatment options, such as granular activated carbon (GAC), membrane, and advanced oxidation processes (AOPs), have shown satisfactory results. The oxidative degradation of some EDCs was associated with aromatic moieties in their structure. Further studies on EDCs need to be conducted, such as source reduction, limiting exposure to vulnerable populations, treatment or remediation of contaminated sites, and the detailed understanding of transport mechanisms in the environment.     

Use of quadrupole time-of-flight mass spectrometry in environmental analysis: elucidation of transformation products of triazine herbicides in water after UV exposure

The high resolution and exact mass capabilities of hybrid quadrupole time-of-flight (QTOF) mass spectrometry can provide an ultimate confirmation in target analysis mode and aid in discovery and elucidation of unknown analytes. In this paper, the latter approach has been applied to study the transformation products of selected pesticides (terbuthylazine, simazine, terbutryn, terbumeton) in environmental waters. Additionally, the usefulness of software modules, originally designed for drug metabolism discovery, has been tested. Different environmental waters spiked at a 0.5 microg/mL level have been irradiated with a mercury lamp for 7 days, sampling 3-mL aliquots every approximately 12 h. After centrifugation, 50 microL was directly injected in an LC-ESI-QTOF system. The high sensitivity in full-scan mode allowed us to elucidate minor metabolites even below 2% of the total peak area. The mass errors observed in almost every case fell below 2 mDa, allowing assignment of a highly probable empirical formula. Besides, the MS/MS capability of this tandem instrument was very useful for differentiation between isomeric transformation products. This work shows that hyphenated LC-QTOF is a powerful approach for the rapid screening and confirmation of unknown pesticide transformation products in environmental water.     

Screening for noncovalent ligand-receptor interactions by electrospray ionization mass spectrometry-based diffusion measurements

The application of a novel method for the identification of low-molecular-weight noncovalent ligands to a macromolecular target is reported. This technique is based on the measurement of analyte diffusion coefficients by electrospray mass spectrometry (ESI-MS) (Clark et al., Rapid Commun. Mass Spectrom. 2002, 16, 1454-1462). Potential ligands have large diffusion coefficients as long as they are free in solution. Binding to a macromolecular target, however, drastically reduces the diffusional mobility of any ligand species. Mixtures containing six different saccharides [ribose, rhamnose, glucose, maltose, maltotriose, and N,N',N'-triacetylchitotriose (NAG(3))] were screened for noncovalent binding to lysozyme. Of these six compounds, only NAG(3) is known to bind to the protein. In "direct" binding tests, NAG(3) shows a significantly reduced diffusion coefficient in the presence of the protein. No changes were observed for any of the other saccharides. In a second set of experiments, the use of a "competition" screening method was explored in which mixtures of candidate saccharides were tested for their ability to displace a reference ligand from the target. The addition of NAG(3)-containing mixtures significantly increased the diffusion coefficient of the reference ligand NAG(4) (N,N',N',N'-tetraacetylchitotetrose), whereas mixtures that did not contain NAG(3) had no effect. These data clearly indicate the potential of ESI-MS-based diffusion measurements as a novel tool to screen compound libraries for binding to proteins and other macromolecular targets. In contrast to conventional ESI-MS-based ligand-receptor binding studies, this method does not rely on the preservation of noncovalent interactions in the gas phase.     

Screening of ion channel receptor agonists using capillary electrophoresis-patch clamp detection with resensitized detector cells

Efficient techniques for identifying endogenous and synthetic ligands of ion channels are important in understanding neuronal communication and for screening drug libraries. This paper describes a technique based on capillary electrophoresis (CE) separation coupled to patch-clamp (PC) detection where a pulsed-flow superfusion scheme was implemented for improved detection. The nicotinic acetylcholine receptor (nAChr) agonists acetylcholine, carbachol, and (-)-nicotine were fractionated and detected by patch-clamped pheochromcytoma detector cells. The high-conductance state of the nAChr during CE-PC detection was maintained and repetitively resensitized using pulsed-flow superfusion with agonist-free buffer. In this way, each agonist evoked an ensemble of peak currents that reflected the spatiotemporal distribution for the ligand at the cell surface. The technique takes advantage of the intrinsic high selectivity and sensitivity of membrane-expressed receptors and allowed for resolution and identification of closely migrating ligands. The method was employed for determination of acetylcholine content in cell lysates.     

Selection of ligands for affinity chromatography using quartz crystal biosensor

This paper described a new strategy for rapid selecting ligands for application in affinity chromatography using a quartz crystal microbalance (QCM) biosensor. An aminoglycoside antibiotic drug, kanamycin (KM), was immobilized on the gold electrodes of the QCM sensor chip. The binding interactions of the immobilized KM with various proteins in solution were monitored as the variations of the resonant frequency of the modified sensor. Such a rapid screen analysis of interactions indicated clearly that KM-immobilized sensor showed strong specific interaction only with lysozyme (LZM). The resultant sensorgrams were rapidly analyzed by using a kinetic analysis software based on a genetic algorithm to derive both the kinetic rate constants (k(ass) and k(diss)) and equilibrium dissociation constants (K(D)) for LZM-KM interactions. The immobilized KM showed higher affinity to LZM with a dissociation constant on the order of 10(-5) M, which is within the range of 10(-4)-10(-8) M and suitable for an affinity ligand. Therefore, KM was demonstrated for the first time as a novel affinity ligand for purification of LZM and immobilized onto the epoxy-activated silica in the presence of a high potassium phosphate concentration. The KM immobilized affinity column has proved useful for a very convenient purification of LZM from chicken egg white. The purity of LZM obtained was higher than 90%, as determined by densitometric scanning of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified fraction. These results confirmed that the selected KM ligand is indeed a valuable affinity ligand for purification of LZM. The new screening strategy based on a QCM biosensor is expected to be a promising way for rapid selecting specific ligands for purifying other valuable proteins.     

Hydroxypyridinone functionalized self-assembled monolayers on nanoporous silica for sequestering lanthanide cations

1,2-Hydroxypyridinone (1,2-HOPO) ligands were installed as self-assembled monolayers on nanoporous silica (MCM-41) to create a superior class of sorbent materials for lanthanide cations. Lanthanides were used as a model system for the radioactive, expensive, and highly hazardous actinides in preliminary screening studies. The ligand properties of the 1,2-HOPO ligand field and the extremely large surface area of MCM-41, coupled with the dense monolayer coating, contribute to the extremely high lanthanide binding capacity of the 1,2-HOPO nanoporous sorbent. At pH 4-5.9, the mass-weighted partition coefficients (Kd) for La, Nd, Eu, and Lu were 354,000, 344,000, 210,800, and 419,800, respectively. The rigid, open pore structure of the silica also allows for very rapid sorption. Being silica-based, the sorbent is compatible with vitrification processing into a final glasseous waste form, for subsequent disposition in a deep geological repository.     

Label-free electrochemical immunoassay for the detection of human chorionic gonadotropin hormone

Here we report on a new and rapid immunoassay for the label-free voltammetric detection of human chorionic gonadotropin hormone (hCG) in urine. Monitoring the changes in the current signals of antibodies (Abs) before and after the binding of the antigen (Ag) provides the basis for an immunoassay that is simple, rapid, and cost-effective. Since hCG is found at highly elevated levels in pregnant female urine with the range of 30,000-200,000 mIU/mL (approximately 30-200 nM) by 8-10 weeks into pregnancy, its label-free electrochemical detection was achieved by using our method. The coverage of the electrode surface with the Ab and the incubation time with the target Ag were optimized for the detection of hCG. The limit of detection of our method was calculated to be 15 pM (n = 3, approximately 15 mIU/mL) in synthetic hCG samples and 20 pM (n = 3, approximately 20 mIU/mL) in human urine. The electrochemical results for the detection of hCG in the urine samples were in agreement with the results obtained using a reference system, enzyme-linked immunosorbent assay. Further research about the intrinsic electroactivity of Abs and their target molecules would surely provide new and sensitive screening assays, as well as extensive data regarding their interaction mechanisms.