Detection Of Human Chorionic Gonadotropin Research Articles

Ultrasensitive electrochemical immunosensor based on Au nanoparticles dotted carbon nanotube–graphene composite and functionalized mesoporous materials

A facile and sensitive electrochemical immunosensor for detection of human chorionic gonadotrophin (hCG) was designed by using functionalized mesoporous nanoparticles as bionanolabels. To construct high-performance electrochemical immunosensor, Au nanoparticles (AuNPs) dotted carbon nanotubes (MWCNTs)–graphene composite was immobilized on the working electrode, which can increase the surface area to capture a large amount of primary antibodies (Ab1) as well as improve the electronic transmission rate. The as-prepared bionanolabels. composed of mesoporous silica nanoparticles (MCM-41) coated with AuNPs through thionine linking, showed good adsorption of horseradish peroxidase-labeled secondary anti-hCG antibody. Interlayer thionine was not only a bridging agent between MCM-41 and AuNPs but also an excellent electron mediator. The approach provided a good linear response range from 0.005 to 500mIUmL−1 with a low detection limit of 0.0026mIUmL−1. The immunosensor showed good precision, acceptable stability and reproducibility. Satisfactory results were obtained for determination of hCG in human serum samples. The proposed method provides a new promising platform of clinical immunoassay for other biomolecules.

Clinical features and microsurgical management of rare tumors in the sellar region

To investigate the clinical characteristics and microsurgical managements of rare tumors in the sellar region. Six rare cases of tumors in the sellar region treated by microsurgery from Jan 2000 to Jan 2010 were reviewed retrospectively. Subsequent treatments were according to the status of preoperative alpha fetal protein (AFP) and human chorionic gonadotropin (HCG) measurement as well as confirmed by histopathological examination in all six patients. Total resection of the tumor was achieved in 2 cases and subtotal resection in 4 cases. Postoperative histopathology confirmed that the lesions were tumors in 5 cases and fungal pseudotumor in 1 case. Moreover, variety of histological types were observed in the present series, including leiomyosarcoma, malignant yolk sac tumor, mixed germ cell tumor, embryonal carcinoma, pilocytic astrocytoma and fungal pseudotumor, respectively. The serum levels of AFP and HCG were elevated to some extent in the patients with malignant yolk sac tumor, mixed germ cell tumor or embryonal carcinoma. Follow-up was conducted in all patients for 1 month to 3 years. The patients with malignant yolk sac tumor and embryonal carcinoma as well as leiomyosarcoma died in 5, 6, 10 months after operation, respectively. Subarachnoid hemorrhage occurred in the case of fungal pseudotumor at 2 months after surgery. The other two patients were surviving well. Rare non-germinomatous malignant germ cell tumors are predominantly susceptible to the sellar region. Furthermore, High misdiagnosis rate and poor prognosis are characteristic in the present study. Dynamic AFP and HCG detection may play an important role in the diagnosis of those non-germinomatous malignant germ cell tumors located in the sellar region. The importance of awareness of the presence of such rare lesions in the sellar region is emphasized.

A Novel Image Methodology for Interpretation of Gold Immunochromatographic Strip

Gold immunochromatographic strip assay is a rapid, simple, single-copy and on-site method. Quantitative Interpretation of the strip can provide more information than the traditional qualitative or semiquantitative strip assay. The paper aims to develop an image based assay method for quantitative determination of trace concentrations by gold immunochromatographic strip. The image of gold immunochromatographic strip is taken by CCD, and, after the proper filter and window cutting, the test line and control line is segmented by the genetic fast fuzzy c-means(FCM) clustering algorithm. In order to improve the measure property, based on Lambert-beer law, the relative reflective integral optical density(RIOD) is selected as the feature by which the interference in the test and control lines can be canceled out each other. The proposed method is applied to the quantitative detection of human chorionic gonadotropin (hCG) as a model. Firstly, the segmentation performance of the genetic fast FCM clustering algorithm is compared with threshold method and FCM clustering algorithm in terms of the peak signal-to-noise ratio (PSNR). Furthermore, the comparison of the blind experiment between the proposed method and commercial quantitative instrument swp-sc1 is carried out. This method is shown to deliver a result comparable and even superior to existing techniques.

Synthesis of size-tunable photoluminescent aqueous CdSe/ZnS microspheres via a phase transfer method with amphiphilic oligomer and their application for detection of HCG antigen

High quality water-soluble photoluminescent (PL) microspheres consisting of CdSe/ZnS quantum dots (QDs) and amphiphilic oligomers (polymaleic acid n-hexadecanol ester) were prepared by a versatile phase transfer method in an emulsion system. Controlled synthesis of different sizes of PL microspheres can be conducted by simply changing the initial oligomer concentration and/or the water/chloroform volume ratio. When the oligomer/QDs molar ratio exceeded 200 : 1, only oligomer-coated monodisperse CdSe/ZnS QDs without any aggregation were obtained. If the molar ratio ranged from 20 : 1 to 120 : 1, size-tunable PL microspheres could be obtained with a size range from 151 to 50 nm. Both of them exhibited high stability in aqueous solution under a wide range of pH, different salt concentrations, and thermal treatment at 100 °C. FTIR spectroscopy, transmission electron microscopy, dynamic light scattering, and fluorescence microscopy were used to characterize the PL microspheres and oligomer-coated monodisperse QDs. It is demonstrated that the stability of PL microspheres indeed depended on their dimensions. Larger PL microspheres could provide more hydrophobic protection for their interior QDs than smaller PL microspheres. A biosensor system (lateral flow immunoassay system, LFIA) for the detection of human chorionic gonadotrophin (HCG) antigen was developed by using CdSe/ZnS PL microspheres as fluorescence labels and a nitrocellulose filter membrane for lateral flow. The result showed that the PL microspheres were excellent fluorescence labels to detect HCG antigen in this LFIA system. The sensitivity of HCG antigen detection can reach 0.5 IU L−1, which is almost 20 times higher than traditional LFIAs using tinctorial labels.

The preparation of label-free electrochemical immunosensor based on the Pt–Au alloy nanotube array for detection of human chorionic gonadotrophin

Background Human chorionic gonadotrophin (hCG) is a secretion of the placenta during pregnancy and gestational trophoblastic diseases. It increases as a consequence of abnormal placental invasion and placental immaturity. So, it is an important diagnostic marker of pregnancy and many diseases such as the hydatidiform mole, the choriocarcinoma, the orchic teratoma, etc. Methods A novel amperometric immunosensor for determination of hCG was constructed by immobilizing hCG antibody on the Pt–Au alloy nanotube array which was produced by electrodepositing strategy using nanopore polycarbonate (PC) membrane at − 0.35 V. The vertically aligned Pt -Au alloy nanotube can be considered as an electrode and therefore each of the electrodes is similar to the nanoelectrode. The determination of hCG antigen was based on its obstruction to the electrocatalytic reduction of H 2O 2 by Pt–Au alloys after binding to the surface of electrode through immunoreactions. Results The electrochemical behaviors were characterized with electrochemical impedance, cyclic voltammetry and chronoamperometry. Under the optimum condition, the current response of the immunosensor is in linear relationship with concentration of hCG ranging from 25 to 400 mIU/ml with a detection limit of 12 mIU/ml. Satisfactory results were obtained for determination of hCG in serum samples. Conclusion This sensor displayed high selectivity, long-term stability and simplicity.

Electrochemically deposited nanocomposite of chitosan and carbon nanotubes for detection of human chorionic gonadotrophin

A new label-free amperometric immunosensor was developed for detection of human chorionic gonadotrophin (hCG) based on multiwall carbon nanotubes–chitosan (MWNTs–CS) complex film and three-dimensional AuNPs–TiO 2 hybrid. Firstly, MWNTs–CS film was deposited on a glassy carbon electrode (GCE) by a simple and controllable electrodeposition method. Next, thionine (Thi), as a redox probe, was covalently bound onto the MWNTs–CS film with glutaraldehyde (GA) to obtain the Thi/MWNTs–CS film. The free amino groups of the composite membrane were used to adsorb AuNPs–TiO 2 for immobilizing human chorionic gonadotrophin antibody (anti-hCG) because of its large surface area and satisfactory biocompatibility. At last, BSA was employed to block possible remaining active sites. Under optimized conditions, the immunosensor displayed high sensitivity, good reproducibility, and a low detection limit of 0.08 mIU/mL at 3 times the background noise. The ease of non-manual technique and the promising feature of biocomposite could serve as a versatile platform for constructing other biosensors.

A new electrochemiluminescence immunosensor based on Ru(bpy) 32+-doped TiO 2 nanoparticles labeling for ultrasensitive detection of human chorionic gonadotrophin

A sensitive electrochemiluminescence (ECL) immunosensor was constructed on the basis of sandwich-type immunoreaction with Nafion functionalized TiO 2 composite nanoparticles as label. The composite nanoparticles labeled antibody were synthesized by successively loading the antibody and large amounts of Ru(bpy) 3 2+ molecules onto the Nafion coated TiO 2 nanoparticles, which is named as Ru–Nafion@TiO 2 labeled secondary antibody. They together with the electrodeposited gold nanoparticles (DpAu) were employed to assemble the ECL immunosensor as following procedure. Firstly, The DpAu was modified on gold electrode to adsorb primary antibody. Then the detected antigen and Ru–Nafion@TiO 2 labeled secondary antibody was conjugated one after another to form a sandwich-type immunocomplex and generate an ECL signal related to the content of analytes. DpAu nanoparticles and composite TiO 2 nanoparticles amplified the ECL response signal via increasing the carrier content of primary antibody and ECL signal labels. The capability of the immunosensor was evaluated by detecting the ECL responses of immunosensor on different concentrations of human chorionic gonadotrophin (HCG). Comparing with other immunoassay, the sandwich-type ECL immunosensor assembled with DpAu and Ru–Nafion@TiO 2 nanoparticles can perform the ultrasensitive detection to HCG with a low detection limit of 0.007 mIU/mL.

Concentration-enhanced rapid detection of human chorionic gonadotropin as a tumor marker using a nanofluidic preconcentrator

Here, we report a new method of concentration-enhanced binding kinetics for a rapid immunoassay screening test on a gold surface in a poly(dimethylsiloxane) (PDMS) microfluidic chip format. The use of alkylthiolate self-assembled monolayers on gold surfaces of a PDMS-glass microchip resulted in accelerated binding kinetics of Human chorionic gonadotropin (hCG) at an electrokinetic trapping zone. We used a PBS solution (buffer concentration ~ 150 mM), not a dibasic buffer system (~10 mM), for the dynamic preconcentrating operation and the preconcentration of cy3 labeled streptavidin onto biotinylated Au surface revealed that the binding kinetics of the protein were linearly proportional to the concentration profile of the preconcentration plug. We showed rapid detection of hCG in the clinical range with a shorten assay time of 10 min. Also, we demonstrated that the amount of sample needed were detection was decreased from ~4 mL to ~25 μL in the standard serum tests. The enhanced binding kinetics between hcG Ag-Ab via preconcentration showed good feasibility for use in a rapid immunoassay screening test.

Detection of Human Chorionic Gonadotropin using Stable Field Effect Transistor with Antibody-Modified Gate

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New discoveries on the biology and detection of human chorionic gonadotropin.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone comprising 2 subunits, alpha and beta joined non covalently. While similar in structure to luteinizing hormone (LH), hCG exists in multiple hormonal and non-endocrine agents, rather than as a single molecule like LH and the other glycoprotein hormones. These are regular hCG, hyperglycosylated hCG and the free beta-subunit of hyperglycosylated hCG.For 88 years regular hCG has been known as a promoter of corpus luteal progesterone production, even though this function only explains 3 weeks of a full gestations production of regular hCG. Research in recent years has explained the full gestational production by demonstration of critical functions in trophoblast differentiation and in fetal nutrition through myometrial spiral artery angiogenesis.While regular hCG is made by fused villous syncytiotrophoblast cells, extravillous invasive cytotrophoblast cells make the variant hyperglycosylated hCG. This variant is an autocrine factor, acting on extravillous invasive cytotrophoblast cells to initiate and control invasion as occurs at implantation of pregnancy and the establishment of hemochorial placentation, and malignancy as occurs in invasive hydatidiform mole and choriocarcinoma. Hyperglycosylated hCG inhibits apoptosis in extravillous invasive cytotrophoblast cells promoting cell invasion, growth and malignancy. Other non-trophoblastic malignancies retro-differentiate and produce a hyperglycosylated free beta-subunit of hCG (hCG free beta). This has been shown to be an autocrine factor antagonizing apoptosis furthering cancer cell growth and malignancy.New applications have been demonstrated for total hCG measurements and detection of the 3 hCG variants in pregnancy detection, monitoring pregnancy outcome, determining risk for Down syndrome fetus, predicting preeclampsia, detecting pituitary hCG, detecting and managing gestational trophoblastic diseases, diagnosing quiescent gestational trophoblastic disease, diagnosing placental site trophoblastic tumor, managing testicular germ cell malignancies, and monitoring other human malignancies. There are very few molecules with such wide and varying functions as regular hCG and its variants, and very few tests with such a wide spectrum of clinical applications as total hCG.

Amperometric immunosensors based on layer-by-layer assembly of gold nanoparticles and methylene blue on thiourea modified glassy carbon electrode for determination of human chorionic gonadotrophin

Nano-Au and methylene blue (MB) were assembled layer-by-layer (LBL) into films on the glassy carbon electrode modified by thiourea for detection of human chorionic gonadotrophin (HCG). The electrode modification process was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electron micrograph (SEM). The factors influencing the performance of the amperometric immunosensor were studied in detail. Tests performed with this immunosensor showed good linearity, the working range for the system was 1.0–100.0 mIU/mL with a detection limit of 0.3 mIU/mL at 3 σ. Moreover, the studied immunosensor exhibited high sensitivity and long-term stability. The present work supplied a promising test method for clinical immunoassay.

Detection of human chorionic gonadotrophin hormone using a label-free epoxysilane-modified capacitive immunosensor

A new and label-free capacitive immunosensor based on antibody-functionalized epoxysilane on a glassy carbon electrode has been developed for quantitative detection of human chorionic gonadotropin (hCG). Monitoring the changes in the capacitance signals of antibodies before and after the binding of the antigen provides the basis for an immunoassay. The performance and factors influencing the immunosensor were also studied. Under the optimized conditions, the developed immunosensor quantitatively detected serum hCG in the range of 18-450 mIU/ml with a detection limit of 5.0 mIU/ml (at 3delta). Thirty-five patients' sera were assayed by the proposed immunosensor, and the results agreed with those given by the commercial radioimmunoassay test kit, with correlation coefficient of 0.998. Further research about the intrinsic electroactivity of antibodies and their target molecules would surely provide new and sensitive screening assays as well as extensive data regarding their interaction mechanisms.

Resin-based micropipette tip for immunochromatographic assays in urine samples

A novel bioanalysis system based on immunochromatography was developed in connection with a nitrocellulose resin modified micropipette tip, such as ZipTip®. The sandwich-type immunoassay was applied to our bioanalysis system. The simple handling of the micropipette enabled us to increase the sample volume and detect low concentrations of target antigens in urine samples. In addition, the washing procedure could also be performed easily to reduce the background signal levels. For analytical evaluations, the color intensity was captured by a flatbed scanner, and processed by a software. We have achieved the detection of human chorionic gonadotropin (hCG) and prostate-specific antigen (PSA). The detection limit of hCG was 0.5 ng/ml (0.05 ng/tip), which is comparable to that of other conventional immunochromatographic systems. Moreover, the detection of PSA was greatly improved over the existing systems with the application of different sample volumes, such as 1 ng/ml (0.2 ng/tip) in a 200 μl sample volume, and 1 ng/ml (0.3 ng/tip) in 300 μl sample volume. Our bioanalysis system is a promising candidate for application to point-of-care tests with its simple handling and high sensitivity.

DNA Directed Protein Immobilization on Mixed ssDNA/Oligo(ethylene glycol) Self-Assembled Monolayers for Sensitive Biosensors

A stable and versatile biosensor surface is prepared by site-directed immobilization of protein-DNA conjugates onto a mixed self-assembled monolayer (SAM) composed of ssDNA thiols and oligo(ethylene glycol) (OEG) terminated thiols. The protein conjugates consist of an antibody chemically linked to a ssDNA target with a sequence complementary to the surface-bound ssDNA probes and are immobilized on the surface via sequence-specific hybridization. Compared to standard antibody immobilization techniques, this approach offers many advantages. The exceptional specificity of DNA hybridization combined with the diversity of potential sequences makes this platform perfect for multichannel sensors. Once a surface is patterned with the appropriate probe sequences, sequence-specific hybridization will sort out the target conjugates and direct them to the appropriate spots on the surface. In addition, the DNA SAMs are very stable and well suited to recycling by dehybdridization of the conjugates from the surface-bound probes. In this work, we demonstrate the specificity, sensitivity, and convenience of using protein-DNA conjugates to convert a DNA/OEG SAM surface into a biosensor surface and apply this platform to the detection of human chorionic gonadotropin using surface plasmon resonance.

Pre- and post-treatment patterns of human chorionic gonadotropin for early detection of persistence after a single dose of methotrexate for ectopic pregnancy

Objective: Given the great variability of human chorionic gonadotropin (HCG) levels after a single dose of methotrexate (MTX) for ectopic pregnancy, it remains complicated to identify cases undergoing persistence until a week from treatment. We evaluated whether pre-treatment patterns of HCG levels could be useful for an earlier detection of persistent trophoblast. Study design: A retrospective study on 62 patients treated by a systemic single dose of methotrexate (50 mg/m 2) for an ectopic pregnancy. Samples for HCG detection were obtained on days −2 and 0 before the therapy, on days +3, +7 and then weekly until values were undetectable. Patients were divided into three groups: Group U (up, meaning “increasing”) and Group D (down, meaning “decreasing”) when HCG levels on day 0 were respectively higher or lower than day −2 level of more than 20% and Group P (plateau) when the difference between day −2 HCG level and the level on day 0 was less than 20%. Results: All the patients of Group D underwent a complete resolution, with a percentage of 33.3% of cases who underwent an initial rise of HCG levels on day +3. The percentage of cases undergoing an initial rise of HCG levels in Group U patients was significantly higher than in Group P patients (60.0% versus 28.6%), but the resolution rate resulted similar in the two groups. For patients of Group P, an increase of HCG levels on day +3 was significantly correlated to the failure of the therapy. Indeed, comparing the cases with an immediate increase of HCG levels to the cases with immediate decrease of HCG levels on day +3, the persistence rate was 80% for the former and 12% for the latter ( P<0.0001). Conclusion: An initial rise of HCG levels after the therapy does not seem to have a clinical relevance in Group D and Group U patients, it well correlates to trophoblastic persistence in Group P patients.

A novel piezoelectric quartz micro-array immunosensor based on self-assembled monolayer for determination of human chorionic gonadotropin

A novel multi-channel 2×5 model of piezoelectric quartz micro-array immunosensor has been developed for quantitative detection of human chorionic gonadotropin (hCG) in serum or urine samples. Every crystal unit of the fabricated piezoelectric hCG micro-array immunosensor can oscillate independently without interfering each other. A 2×5 model of micro-array immunosensor as compared with a one-channel immunosensor can provide eight times higher detection speeds for hCG assay. The anti-hCG antibody is deposited on the gold electrode’s surface of 10 MHz quartz AT-cut crystal by self-assembled technique using sulfosuccinimidyl 6-[3′-(2-pyridyldithio) propionamido] hexanoate (Sulfo-LC-SPDP), and serves as an antibody recognizing layer. The highly ordered self-assembled monolayers (SAM) ensure well-controlled surface structure and offer many advantages to the performance of the sensor. Compared with conventional antibody immobilization methods, the amount and the reaction activity of antibody monolayer coated by the SAM binding are bigger than those by the SPA method, and less non-specific binding caused by other analytes in sample is found. Under the optimized experimental conditions, the results showed that micro-array immunosensor quantitatively detected serum or urine hCG in the range of 2.5–500 mIU/ml with high precision (CV<5%); other hormones in human serum and urine did not interfere with the determination markedly. Serum and urine samples of 60 patients were detected by the micro-array immunosensor, and the results agreed well with those given by the commercial radioimmunoassay test kit, with correlation coefficient of 0.92. After regeneration with urea solution the coated immunosensor can be reused five times without appreciable loss of activity.

Human chorionic gonadotropin patterns after a single dose of methotrexate for ectopic pregnancy

Objective: The great variability in human chorionic gonadotropin (HCG) levels after a single dose of methotrexate (MTX) for ectopic pregnancy makes it difficult to predict treatment failure. We describe different patterns of HCG levels. Study design: Fifty patients were injected i.m. with 50mg/m2 of MTX for an ectopic pregnancy. Venous blood samples for HCG detection were obtained on the day of treatment (day 0), day 3 and day 7 and weekly until values were undetectable. Patients were classified as: group 1, persistent pathology (n=11); group 2, complete resolution with a decrease of HCG levels at day 3 (n=30); group 3, complete resolution after a rise of HCG values at day 3 (n=9). Statistical analysis was performed using the Mann–Whitney non-parametric test with 95% confidence intervals. Results: Values of day 0 were similar for all the groups. HCG levels of group 3 decreased rapidly after day 3 and at day 7 they were significantly different from levels of group 1. Differences in HCG levels between groups 2 and 3 became indistinguishable from day 21. Conclusion: The observation of patients undergoing resolution after an initial increase of HCG levels justify an expectant management for 1 week in clinically stable patients. The strategy to separate HCG curves in patients undergoing resolution may shed light on the different clinical responses to therapy for ectopic pregnancies. However, the phenomenon of the immediate rise of HCG should be better investigated.

Construction of electrochemical flow immunoassay system using capillary columns and ferrocene conjugated immunoglobulin G for detection of human chorionic gonadotrophin

In this paper is reported a miniaturized flow immunoassay system. Ferrocenecarboxylic acid (Fc) conjugated with anti-HCG immunoglobulin G (IgG) antibody (Fc–IgG) was prepared, and used as a novel analytical reagent. The system consists of the immunoreaction section, the capillary column packed with cation exchange resin, and the flow cell for electrochemical detection of Fc–IgG. Antibody–antigen complexes were separated from their free conjugate on the basis of differences in isoelectric point (pI) using a cation exchange capillary column. The assay yielded a linear relationship between signal and HCG concentration in the range 0–2000 mIU/ml. This simple technique enables the assay of HCG within 2 min. The cation exchange capillary column was regenerated by occasional elution with malonate buffer (pH 6.0) containing 0.5 M NaCl, to remove free conjugate. Free conjugate recovered in this manner could be reused up to eight times without significant decreases in the sensitivity of the immunoassay. This electrochemical flow immunoassay requires only minute quantities of serum and generates highly reproducible results.

Flow immunoassay for detection of human chorionic gonadotrophin using a cation exchange resin packed capillary column

A human chorionic gonadotrophin (HCG) detection was carried out by the flow immunoassay system with a cation exchange resin packed capillary column (762 μm i.d.×10 cm length), that can be considered a miniaturised version of the previously reported system. The HCG and fluorescein isothiocyanate (FITC) conjugated immunoglobulin G (IgG) antibody complex was separated from free FITC conjugated IgG on the basis of differences in isoelectric points. The fluorescence intensity correlated linearly with concentrations of HCG in the range of 25–1500 mIU/ml. This simple technique permits the assay of HCG within 1.3 min. It was possible to reuse of free FITC conjugated IgG. This simple and rapid immunoassay method can be used for the continuous detection of HCG.

An indirect perfluorosulfonated ionomer-coated electrochemical immunosensor for the detection of the protein human chorionic gonadotrophin.

The development of an amperometric immunosensor for the detection of human chorionic gonadotrophin (hCG) is described. In this immunosensor, Nafion was used to immobilize an anti-hCG monoclonal antibody onto a glassy carbon electrode. A systematic study on the effects of experimental parameters such as the quantity of ethanol present in the Nafion solution, the percentage composition of Nafion, the pH of the immobilization buffer, and the concentration of antibody used for entrapment experiments on the binding between the immobilized antibody and 125I-labeled hCG has been carried out. Two immobilization methods, coimmobilization and adsorption immobilization, have then been attempted. A binding of approximately 3% was obtained in the former method, while 5.5% binding was achieved in the latter. On the basis of these results, adsorption immobilization was employed to entrap antibody on the electrode surface. A sandwich assay was then developed for hCG in which the enzyme horseradish peroxidase was conjugated to a second anti-hCG monoclonal antibody. The activity of the enzyme was determined electrochemically by the reduction of benzoquinone to hydroquinone. Binding of hCG to immobilized antibody determines the quantity of enzyme-conjugated antibody at the electrode surface, permitting the quantification of hCG. By a standard additions calibration method of hCG performed in blank human serum samples, the immunosensor exhibits a limit of linearity at 200 mIU mL-1 and a detection limit of 11.2 mIU mL-1 (based on twice the standard deviation of the blank solution).