Amplification of antigen-antibody interactions based on biotin labeled protein-streptavidin network complex using impedance spectroscopy

Antibody was covalently immobilized by amine coupling method to gold surfaces modified with a self-assembled monolayer of thioctic acid. The electrochemical measurements of cyclic voltammetry and impedance spectroscopy showed that the hexacyanoferrate redox reactions on the gold surface were blocked due to the procedures of self-assembly of thioctic acid and antibody immobilization. The binding of a specific antigen to antibody recognition layer could be detected by measurements of the impedance change. A new amplification strategy was introduced for improving the sensitivity of impedance measurements using biotin labeled protein- streptavidin network complex. This amplification strategy is based on the construction of a molecular complex between streptavidin and biotin labeled protein. This complex can be formed in a cross-linking network of molecules so that the amplification of response signal will be realized due to the big molecular size of complex. The results show that this amplification strategy causes dramatic improvement of the detection sensitivity of hIgG and has good correlation for detection of hIgG in the range of 2-10 mug/ml. (C) 2001 Elsevier Science B.V. All rights reserved.

Electrochemical biosensor based on microfabricated electrode arrays for life sciences applications

In developing a biosensor, the utmost important aspects that need to be emphasized are the specificity and selectivity of the transducer. These two vital prerequisites are of paramount in ensuring a robust and reliable biosensor. Improvements in electrochemical sensors can be achieved by using microelectrodes and to modify the electrode surface (using chemical or biological recognition layers to improve the sensitivity and selectivity). The fabrication and characterisations of silicon-based and glass-based gold microelectrode arrays with various geometries (band and disc) and dimension (ranging from 10 μm-100 nm) were reported. It was found that silicon-based transducers of 10 μm gold microelectrode array exhibited the most stable and reproducible electrochemical measurements hence this dimension was selected for further study. Chemical electrodeposition on both 10 μm microband and microdisc were found viable by electro-assisted self-assembled sol-gel silica film and nanoporous-gold electrodeposition respectively. The fabrication and characterisations of on-chip electrochemical cell was also reported with a fixed diameter/width dimension and interspacing variation. With this regard, the 10 μm microelectrode array with interspacing distance of 100 μm exhibited the best electrochemical response. Surface functionalisations on single chip of planar gold macroelectrodes were also studied for the immobilisation of histidine-tagged protein and antibody. Imaging techniques such as atomic force microscopy, fluorescent microscopy or scanning electron microscope were employed to complement the electrochemical characterisations. The long-chain thiol of self-assembled monolayer with NTA-metal ligand coordination was selected for the histidine-tagged protein while silanisation technique was selected for the antibody immobilisation. The final part of the thesis described the development of a T-2 labelless immunosensor using impedimetric approach. Good antibody calibration curve was obtained for both 10 μm microband and 10 μm microdisc array. For the establishment of the T-2/HT-2 toxin calibration curve, it was found that larger microdisc array dimension was required to produce better calibration curve. The calibration curves established in buffer solution show that the microelectrode arrays were sensitive and able to detect levels of T-2/HT-2 toxin as low as 25 ppb (25 μg kg-1) with a limit of quantitation of 4.89 ppb for a 10 μm microband array and 1.53 ppb for the 40 μm microdisc array.

Strategies to Improve the Surface Plasmon Resonance-Based Immmunodetection of Bacterial Cells

We have made a comparison of (a) different surface chemistries of surface plasmon resonance (SPR) sensor chips (such as carboxymethylated dextran and carboxymethylated C1) and (b) of different assay formats (direct, sandwich and subtractive immunoassay) in order to improve the sensitivity of the determination of the model bacteria Acidovorax avenae subsp. citrulli (Aac). The use of the carboxymethylated sensor chip C1 resulted in a better sensitivity than that of carboxymethylated dextran CM5 in all the assay formats. The direct assay format, in turn, exhibits the best sensitivity. Thus, the combination of a carboxymethylated sensor chip C1 with the direct assay format resulted in the highest sensitivity for Aac, with a limit of detection of 1.6x106 CFU mL-1. This SPR immunosensor was applied to the detection of Aac in watermelon leaf extracts spiked with the bacteria, and the lower LOD is 2.2x107 CFU mL-1.

Detection of Tetrodotoxins in Puffer Fish by a Self-Assembled Monolayer-Based Immunoassay and Comparison with Surface Plasmon Resonance, LC-MS/MS, and Mouse Bioassay

The increasing occurrence of puffer fish containing tetrodotoxin (TTX) in the Mediterranean could represent a major food safety risk for European consumers and threaten the fishing industry. The work presented herein describes the development of a new enzyme linked immunosorbent assay (mELISA) based on the immobilization of TTX through dithiol monolayers self-assembled on maleimide plates, which provides an ordered and oriented antigen immobilization and favors the antigen-antibody affinity interaction. The mELISA was found to have a limit of detection (LOD) of TTX of 0.23 mg/kg of puffer fish matrix. The mELISA and a surface plasmon resonance (SPR) immunosensor previously developed were employed to establish the cross-reactivity factors (CRFs) of 5,6,11-trideoxy-TTX, 5,11-deoxy-TTX, 11-nor-TTX-6-ol, and 5,6,11-trideoxy-4-anhydro-TTX, as well as to determine TTX equivalent contents in puffer fish samples. Results obtained by both immunochemical tools were correlated (R(2) = 0.977). The puffer fish samples were also analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the corresponding CRFs were applied to the individual TTX contents. Results provided by the immunochemical tools, when compared with those obtained by LC-MS/MS, showed a good degree of correlation (R(2) = 0.991 and 0.979 for mELISA and SPR, respectively). The mouse bioassay (MBA) slightly overestimated the CRF adjusted TTX content of samples when compared with the data obtained from the other techniques. The mELISA has been demonstrated to be fit for the purpose for screening samples in monitoring programs and in research activities.

A potentiometric biosensor for rapid on-site disease diagnostics

Quantitative point-of-care (POC) devices are the next generation for serological disease diagnosis. Whilst pathogen serology is typically performed by centralized laboratories using Enzyme-Linked ImmunoSorbent Assay (ELISA), faster on-site diagnosis would infer improved disease management and treatment decisions. Using the model pathogen Bovine Herpes Virus-1 (BHV-1) this study employs an extended-gate field-effect transistor (FET) for direct potentiometric serological diagnosis. BHV-1 is a major viral pathogen of Bovine Respiratory Disease (BRD), the leading cause of economic loss ($2 billion annually in the US only) to the cattle and dairy industry. To demonstrate the sensor capabilities as a diagnostic tool, BHV-1 viral protein gE was expressed and immobilized on the sensor surface to serve as a capture antigen for a BHV-1-specific antibody (anti-gE), produced in cattle in response to viral infection. The gE-coated immunosensor was shown to be highly sensitive and selective to anti-gE present in commercially available anti-BHV-1 antiserum and in real serum samples from cattle with results being in excellent agreement with Surface Plasmon Resonance (SPR) and ELISA. The FET sensor is significantly faster than ELISA (<10 min), a crucial factor for successful disease intervention. This sensor technology is versatile, amenable to multiplexing, easily integrated to POC devices, and has the potential to impact a wide range of human and animal diseases.

Imunossensores Eletroquímicos para o Diagnóstico Precoce e Descentralizado do Cancro da Mama

O cancro é uma das principais causas de morte em todo o mundo. Entre as mulheres, o cancro da mama é o mais frequente. A deteção precoce do cancro é de extrema importância na medida em que pode aumentar as possibilidades de cura dos pacientes e contribuir para a diminuição da taxa de mortalidade desta doença. Um método que tem contribuído para a deteção precoce do cancro é a análise de biomarcadores. Biomarcadores associados ao cancro da mama, como o Recetor 2 do Fator de Crescimento Epidérmico Humano (HER2) e o Antigénio Carbohidratado 15-3 (CA 15-3), podem ser detetados através de dispositivos como os biossensores. Neste trabalho foram desenvolvidos dois imunossensores eletroquímicos para a análise de HER2 e CA 15-3. Para ambos os sensores foram utilizados, como transdutores, elétrodos serigrafados de carbono. A superfície destes transdutores foi nanoestruturada com nanopartículas de ouro. Foram realizados imunoensaios não-competitivos (do tipo sandwich) em ambos os imunossensores, cuja estratégia consistiu na (i) imobilização do respetivo anticorpo de captura na superfície nanoestruturada dos elétrodos, (ii) bloqueio da superfície com caseína, (iii) incubação com uma mistura do analito (HER2 ou CA 15-3) e o respetivo anticorpo de deteção biotinilado, (iv) adição de estreptavidina conjugada com fosfatase alcalina (S-AP; a AP foi utilizada como marcador enzimático), (v) adição de uma mistura do substrato enzimático (3-indoxil fosfato) e nitrato de prata, e (vi) deteção do sinal analítico através da redissolução anódica, por voltametria de varrimento linear, da prata depositada enzimaticamente. Com as condições experimentais otimizadas, foi estabelecida a curva de calibração para a análise de HER2 em soro, entre 15 e 100 ng/mL, obtendo-se um limite de deteção de 4,4 ng/mL. Para o CA 15-3 a curva de calibração (em solução aquosa) foi estabelecida entre 15 e 250 U/mL, obtendo-se um limite de deteção de 37,5 U/mL. Tendo em conta o valor limite (cutoff value) estabelecido para o HER2 (15 ng/mL) pode-se comprovar a possível utilidade do imunossensor desenvolvido para o diagnóstico precoce e descentralizado do cancro da mama. No caso do CA 15-3 serão necessários estudos adicionais para se poder avaliar a utilidade do imunossensor para o diagnóstico do cancro da mama.

Chitosan/AuNPs Modified Graphene Electrochemical Sensor for Label-Free Human Chorionic Gonadotropin Detection

A new immunosensor is presented for human chorionic gonadotropin (hCG), made by electrodepositing chitosan/gold-nanoparticles over graphene screen-printed electrode (SPE). The antibody was covalently bound to CS via its Fc-terminal. The assembly was controlled by electrochemical Impedance Spectroscopy (EIS) and followed by Fourier Transformed Infrared (FTIR). The hCG-immunosensor displayed linear response against the logarithm-hCG concentration for 0.1–25 ng/mL with limit of detection of 0.016 ng/mL. High selectivity was observed in blank urine and successful detection of hCG was also achieved in spiked samples of real urine from pregnant woman. The immunosensor showed good detection capability, simplicity of fabrication, low-cost, high sensitivity and selectivity.

Label-free human chorionic gonadotropin detection at picogram levels using oriented antibodies bound to graphene screen-printed electrodes

Human chorionic gonadotropin (hCG) is a key diagnostic marker of pregnancy and an important biomarker for cancers in the prostate, ovaries and bladder and therefore of great importance in diagnosis. For this purpose, a new immunosensor of screen-printed electrodes (SPEs) is presented here. The device was fabricated by introducing a polyaniline (PANI) conductive layer, via in situ electropolymerization of aniline, onto a screen-printed graphene support. The PANI-coated graphene acts as the working electrode of a three terminal electrochemical sensor. The working electrode is functionalised with anti-hCG, by means of a simple process that enabled oriented antibody binding to the PANI layer. The antibody was attached to PANI following activation of the –COOH group at the Fc terminal. Functionalisation of the electrode was analysed and optimized using Electrochemical Impedance Spectroscopy (EIS). Chemical modification of the surface was characterised using Fourier transform infrared, and Raman spectroscopy with confocal microscopy. The graphene–SPE–PANI devices displayed linear responses to hCG in EIS assays from 0.001 to 50 ng mL−1 in real urine, with a detection limit of 0.286 pg mL−1. High selectivity was observed with respect to the presence of the constituent components of urine (urea, creatinine, magnesium chloride, calcium chloride, sodium dihydrogen phosphate, ammonium chloride, potassium sulphate and sodium chloride) at their normal levels, with a negligible sensor response to these chemicals. Successful detection of hCG was also achieved in spiked samples of real urine from a pregnant woman. The immunosensor developed is a promising tool for point-of-care detection of hCG, due to its excellent detection capability, simplicity of fabrication, low-cost, high sensitivity and selectivity.

Detection of the peanut allergen Ara h 6 in foodstuffs using a voltammetric biosensing approach

A voltammetric biosensor for Ara h 6 (a peanut allergen) detection in food samples was developed. Gold nanoparticle-modified screen-printed carbon electrodes were used to develop a sandwich-type immunoassay using two-monoclonal antibodies. The antibody-antigen interaction was detected through the electrochemical detection of enzymatically deposited silver. The immunosensor presented a linear range between 1 and 100 ng/ml, as well as high precision (inter-day RSD ≤9.8 %) and accuracy (recoveries ≥96.7 %). The detection and quantification limits were 0.27 and 0.88 ng/ml, respectively. It was possible to detect small levels of Ara h 6 in complex food matrices.

Investigation of the interaction between Tc85-11 protein and antibody anti-T cruzi by AFM and amperometric measurements

This present work reports on development of an amperometric immunosensor for the diagnosis of Chagas' disease using a specific glycoprotein of the trypomastigote surface, which belongs to the Tc85-11 protein family of Trypanosoma cruzi (T cruzi). An atomically flat gold surface on a silicon substrate and gold screen-printed electrodes were functionalized with cystatrine and later activated with glutaraldehyde (GA), which was used to form covalent bonds with the purified recombinant antigen (Tc85-11). The antigen reacts with the antibody from the serum, and the affinity reaction was monitored directly using atomic force microscopy or amperometry through a secondary antibody tagged to peroxidase (HRP). Surface imaging allowed to us to differentiate the modification steps and antigen-antibody interaction allowed to distinguish the affinity reactions. In the amperometric immunosensor, peroxidase catalyses the L-2 formation in the presence of hydrogen peroxide and potassium iodide, and the reduction current intensity was measured at a given potential with screen-printed electrodes. The immunosensor was applied to sera of chagasic patients and patients having different systemic diseases. (c) 2006 Elsevier Ltd. All rights reserved.

Imunossensor amperométrico

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Magneto Immunoassays for Plasmodium falciparum Histidine-Rich Protein 2 Related to Malaria based on Magnetic Nanoparticles

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A disposable chitosan-modified carbon fiber electrode for dengue virus envelope protein detection

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

New label free CA125 detection based on gold nanostructured screen-printed electrode

This paper describes the optimisation and the analytical performances of a label-free impedimetric immunosensor for the detection of tumour marker CA125 based on gold nanoparticles modified screen-printed graphite electrode. Experimental conditions of each step for the developed immunosensor were studied and optimised. The immunosensor response varied linearly (r2 = 0.996) with antigen concentration between 0 and 100 U/mL. The estimated detection limit was 6.7 U/mL. The electrochemical immunosensor allowed unambiguous identification of CA125, while no significant non-specific signal was detected in the case of all negative controls. The analytical usefulness of the impedimetric immunosensor was finally demonstrated analysing serum samples. © 2012 Elsevier B.V. All rights reserved.

Label free redox capacitive biosensing

A surface confined redox group contributes to an interfacial charging (quantifiable by redox capacitance) that can be sensitively probed by impedance derived capacitance spectroscopy. In generating mixed molecular films comprising such redox groups, together with specific recognition elements (here antibodies), this charging signal is able to sensitively transduce the recognition and binding of specific analytes. This novel transduction method, exemplified here with C-reactive protein, an important biomarker of cardiac status and general trauma, is equally applicable to any suitably prepared interfacial combination of redox reporter and receptor. The assays are label free, ultrasensitive, highly specific and accompanied by a good linear range. © 2013 Elsevier B.V.