Immunosensors based on layer-by-layer self-assembled Au colloidal electrode for the electrochemical detection of antigen

Colloidal Au particles have been deposited on the gold electrode through layer-by-layer self-assembly using cysteamine as cross-linkers. Self-assembly of colloidal Au on the gold electrode resulted in ail easier attachment of antibody, larger electrode surface and ideal electrode behavior. The redox reactions of [Fe(CN)(6)]-/[Fe(CN)(6)](3-) on the gold surface were blocked due to antibody immobilization, which were investigated by cyclic voltammetry and impedance spectroscopy. The interaction of antigen with grafted antibody recognition layers was carried out by soaking the modified electrode into a phosphate buffer at pH 7.0 with various concentrations of antigen at 37degreesC for 30 min. Further, an amplification strategy to use biotin conjugated antibody was introduced for improving the sensitivity of impedance measurements. Thus, the sensor based oil this immobilization method exhibits a large linear dynamic range, from 5 - 400 mug/L for detection of Human IgG. The detection limit is about 0.5 mug/L.

The progress in the method of immobilizing biomolecules for piezoelectric immunosensors

The working principle of piezoelectric mass sensor is generally introduced. Tbe recent progress about the method of immobilizing biomolecule, such as antigen, antibody etc. onto piezoelectric crystal surfaces has been reported, including the way of directly immobilizing biomolecules, and immobilizing them using protein A(or protein G), polymer, silianizition agent, SAM technique, LB monolayer technique etc.. At last, some recent trends of the field has been outlined.

Development of micro immunosensors to study genomic and proteomic biomarkers related to cancer and Alzheimer's disease

A report from the National Institutes of Health defines a disease biomarker as a “characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.” Early diagnosis is a crucial factor for incurable disease such as cancer and Alzheimer’s disease (AD). During the last decade researchers have discovered that biochemical changes caused by a disease can be detected considerably earlier as compared to physical manifestations/symptoms. In this dissertation electrochemical detection was utilized as the detection strategy as it offers high sensitivity/specificity, ease of operation, and capability of miniaturization and multiplexed detection. Electrochemical detection of biological analytes is an established field, and has matured at a rapid pace during the last 50 years and adapted itself to advances in micro/nanofabrication procedures. Carbon fiber microelectrodes were utilized as the platform sensor due to their high signal to noise ratio, ease and low-cost of fabrication, biocompatibility, and active carbon surface which allows conjugation with biorecognition moieties. This dissertation specifically focuses on the detection of 3 extensively validated biomarkers for cancer and AD. Firstly, vascular endothelial growth factor (VEGF) a cancer biomarker was detected using a one-step, reagentless immunosensing strategy. The immunosensing strategy allowed a rapid and sensitive means of VEGF detection with a detection limit of about 38 pg/mL with a linear dynamic range of 0–100 pg/mL. Direct detection of AD-related biomarker amyloid beta (Aβ) was achieved by exploiting its inherent electroactivity. The quantification of the ratio of Aβ1-40/42 (or Aβ ratio) has been established as a reliable test to diagnose AD through human clinical trials. Triple barrel carbon fiber microelectrodes were used to simultaneously detect Aβ1-40 and Aβ1-42 in cerebrospinal fluid from rats within a detection range of 100nM to 1.2μM and 400nM to 1μM respectively. In addition, the release of DNA damage/repair biomarker 8-hydroxydeoxyguanine (8-OHdG) under the influence of reactive oxidative stress from single lung endothelial cell was monitored using an activated carbon fiber microelectrode. The sensor was used to test the influence of nicotine, which is one of the most biologically active chemicals present in cigarette smoke and smokeless tobacco.

Photoresist functionalisation method for high-density protein microarrays using photolithography

Since the last decade, there is a growing need for patterned biomolecules for various applications ranging from diagnostic devices to enabling fundamental biological studies with high throughput. Protein arrays facilitate the study of protein-protein, protein-drug or protein-DNA interactions as well as highly multiplexed immunosensors based on antibody-antigen recognition. Protein microarrays are typically fabricated using piezoelectric inkjet printing with resolution limit of similar to 70-100 mu m limiting the array density. A considerable amount of research has been done on patterning biomolecules using customised biocompatible photoresists. Here, a simple photolithographic process for fabricating protein microarrays on a commercially available diazo-naphthoquinone-novolac-positive tone photoresist functionalised with 3-aminopropyltriethoxysilane is presented. The authors demonstrate that proteins immobilised using this procedure retain their activity and therefore form functional microarrays with the array density limited only by the resolution of lithography, which is more than an order of magnitude compared with inkjet printing. The process described here may be useful in the integration of conventional semiconductor manufacturing processes with biomaterials relevant for the creation of next-generation bio-chips.

Synthesis and electrochemical applications of gold nanoparticles

This review covers recent advances in synthesis and electrochemical applications of gold nanoparticles (AuNPs). Described approaches include the synthesis of AuNPs via designing and choosing new protecting ligands; and applications in electrochemistry of AuNPs including AuNPs-based bioelectrochemical sensors, such as direct electrochemistry of redox-proteins, genosensors and immunosensors, and AuNPs as enhancing platform for electrocatalysis and electrochemical sensors.

Thin film composite optical waveguides for sensor applications: a review

We review the design and fabrication of thin-film composite optical waveguides (OWG) with high refractive index for sensor applications. A highly sensitive optical sensor device has been developed on the basis of thin-film, composite OWG. The thin-film OWG was deposited onto the surface of a potassium-ion-exchanged (K+) glass OWG by sputtering or spin coating (5-9 mm wide, and with tapers at both ends). By allowing an adiabatic transition of the guided light from the secondary OWG to the thin-film OWG, the electric field of the evanescent wave at the thin film was enhanced. The attenuation of the guided light in the thin film layer was small, and the guided light intensity changed sensitively with the refractive index of the cladding layer. Our experimental results demonstrate that thin-film, composite OWG gas sensors or immunosensors are much more sensitive than sensors based on other technologies. (c) 2004 Elsevier B.V. All rights reserved.

A new method for non-labeling attomolar detection of diseases based on an individual gold nanorod immunosensor

Herein, we present the use of a single gold nanorod sensor for detection of diseases on an antibody-functionalized surface, based on antibody–antigen interaction and the localized surface plasmon resonance (LSPR) ?max shifts of the resonant Rayleigh light scattering spectra. By replacing the cetyltrimethylammonium bromide (CTAB), a tightly packed self-assembled monolayer of HS(CH2)11(OCH2CH2)6OCH2COOH(OEG6) has been successfully formed on the gold nanorod surface prior to the LSPR sensing, leading to the successful fabrication of individual gold nanorod immunosensors. Using prostate specific antigen (PSA) as a protein biomarker, the lowest concentration experimentally detected was as low as 111 aM, corresponding to a 2.79 nm LSPR ?max shift. These results indicate that the detection platform is very sensitive and outperforms detection limits of commercial tests for PSA so far. Correlatively, its detection limit can be equally compared to the assays based on DNA biobarcodes. This study shows that a gold nanorod has been used as a single nanobiosensor to detect antigens for the first time; and the detection method based on the resonant Rayleigh scattering spectrum of individual gold nanorods enables a simple, label-free detection with ultrahigh sensitivity.

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.

Electrochemical immunosensor for multiplexed detection of food-borne pathogens using nanocrystal bioconjugates and MWCNT screen-printed electrode

Bacterial food poisoning is an ever-present threat that can be prevented with proper care and handling of food products. A disposable electrochemical immunosensor for the simultaneous measurements of common food pathogenic bacteria namely Escherichia coli O157:H7 (E. coli), campylobacter and salmonella were developed. The immunosensor was fabricated by immobilizing the mixture of anti-E. coli, anticampylobacter and anti-salmonella antibodies with a ratio of 1:1:1 on the surface of the multiwall carbon nanotube-polyallylamine modified screen printed electrode (MWCNT-PAH/SPE). Bacteria suspension became attached to the immobilized antibodies when the immunosensor was incubated in liquid samples. The sandwich immunoassay was performed with three antibodies conjugated with specific nanocrystal ( -E. coli-CdS, -campylobacter-PbS and -salmonella-CuS) which has releasable metal ions for electrochemical measurements. The square wave anodic stripping voltammetry (SWASV) was employed to measure released metal ions from bound antibody nanocrystal conjugates. The calibration curves for three selected bacteria were found in the range of 1 × 103 – 5 × 105 cells mL−1 with the limit of detection (LOD) 400 cells mL−1 for salmonella, 400 cells mL−1 for campylobacter and 800 cells mL−1 for E. coli. The precision and sensitivity of this method show the feasibility of multiplexed determination of bacteria in milk samples.

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.

Double-Tagging Polymerase Chain Reaction with a Thiolated Primer and Electrochemical Genosensing based on Gold Nanocomposite Sensor for Food Safety

A novel material for electrochemical biosensing based on rigid conducting gold nanocomposite (nano-AuGEC) is presented. Islands of chemisorbing material (gold nanoparticles) surrounded by nonreactive, rigid, and conducting graphite epoxy composite are thus achieved to avoid the stringent control of surface coverage parameters required during immobilization of thiolated oligos in continuous gold surfaces. The spatial resolution of the immobilized thiolated DNA was easily controlled by merely varying the percentage of gold nanoparticles in the composition of the composite. As low as 9 fmol (60 pM) of synthetic DNA were detected in hybridization experiments when using a thiolated probe. Moreover, for the first time a double tagging PCR strategy was performed with a thiolated primer for the detection of Salmonella sp., one of the most important foodborne pathogens affecting food safety. Ibis assay was performed by double-labeling the amplicon during the PCR with a -DIG and -SH set of labeled primers. The thiolated end allows the immobilization of the amplicon on the nano-AuGEC electrode, while digoxigenin allows the electrochemical detection with the antiDIG-HRP reporter in the femtomole range. Rigid conducting gold nanocomposite represents a good material for the improved and oriented immobilization of biomolecules with excellent transducing properties for the construction of a wide range of electrochemical biosensors such as immunosensors, genosensors, and enzymosensors.

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

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Disposable immunosensor for human cardiac troponin T based on streptavidin-microsphere modified screen-printed electrode

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.