Impedimetric biosensor for cancer cell detection

Electrochemical impedance spectroscopy was evaluated for the label free detection of MCF-7 cancer cell in which c-erbB-2 receptor is overexpressed on the cell surfaces. Anti-c-erbB-2, used as a specific antibody, was immobilized on electrogenerated polypyrrole-NHS on electrodes via covalent linking. The polymer formation, the grafting of the antibody, and the recognition event with the cancer cells using MCF-7 as a model cell line, were characterized by using cyclic voltammetry and fluorescence microscopy. The impedimetric sensor showed high sensitivity from 100 to 10 000 cell/mL without needing any labeling step and represents an efficient transduction method for cell selective detection.     

Nanostructured zirconium oxide based genosensor for Escherichia coli detection

A deoxyribonucleic acid (DNA) biosensor has been fabricated via immobilization of 17 base terminal single stranded DNA (ssDNA) identified from the 16s rRNA coding region of Escherichia coli onto sol–gel derived nanostructured zirconium oxide (NanoZrO₂) film. An oligonucleotide probe with a terminal 5′-phosphate group has been attached to the surface of the electrode via affinity of NanoZrO₂ for phosphate. The results of hybridization studies carried out with the complementary, non-complementary and genomic DNA reveal that ssDNA/NanoZrO₂/ITO bioelectrode has a high selectivity and sensitivity towards hybridization detection with limits of 10^?6–10⁶ pM of complementary DNA.     

Development of electrochemical genosensor for MYCN oncogene detection using rhodamine B as electroactive label

A novel electrochemical genosensor based on a graphite electrode modified with poly(4-aminophenol) has been constructed for prognostic of neuroblastoma, a malignant tumor originating from embryonic precursor cells of the sympathetic nervous system and associated with the amplification of the MYCN oncogene. The genosensor exhibited distinct electrical and morphological properties using rhodamine B as indicator of DNA hybridization. The detection limit was evaluated to be 0.47 μmol L^?1 (n = 3), and the electrochemical genosensor was selective for the complementary DNA, using serum sample. This DNA sensing platform was successfully applied to detect MYCN, an important biomarker for neuroblastoma.     

Microfluidic-based electrochemical genosensor coupled to magnetic beads for hybridization detection

This paper describes the development of a rapid and sensitive enzyme-linked electrochemical genosensor using a novel microfluidic-based platform. In this work, hybridization was performed on streptavidin-coated paramagnetic micro-beads functionalized with a biotinylated capture probe. The complementary sequence was then recognized via sandwich hybridization with a capture probe and a biotinylated signaling probe. After labeling the biotinylated hybrid with a streptavidin-alkaline phosphatase conjugate, the beads were introduced in a disposable cartridge composed of eight parallel microchannels etched in a polyimide substrate. The modified beads were trapped with a magnet addressing each microchannel individually. The presence of microelectrodes in each channel allowed direct electrochemical detection of the enzymatic product within the microchannel. Detection was performed in parallel within the eight microchannels, giving rise to the possibility of performing a multiparameter assay. Quantitative determinations of the analyte concentrations were obtained by following the kinetics of the enzymatic reaction in each channel. The chip was regenerated after each assay by removing the magnet and thus releasing the magnetic beads. The system was applied to the analytical detection of PCR amplified samples with a RSD% = 6. A detection limit of 0.2 nM was evaluated.     

Impedimetric genosensors for the detection of DNA hybridization

Impedance spectroscopy is proposed as the transduction principle for detecting the hybridization of DNA complementary strands. In our experiments, different DNA oligonucleotides were used as model gene substances. The gene probe is first immobilized on a graphite-epoxy composite working electrode based genosensor. Detection principle is based on changes of impedance spectra of a redox marker, the ferro/ferricyanide couple, after hybridization with target DNA. Resistance offered to the electrochemical reaction serves as the working signal, allowing for an unlabelled gene assay.      

Impedimetric biosensor for early detection of cervical cancer

An impedimetric biosensor based on PEGylated arginine functionalized magnetic nanoparticles for early detection of cervical cancer is reported. The cervical cancer cells could be selectively and sensitively detected down to 10 cells mL(-1) on the modified electrode, which is promising for advancement in clinical diagnosis and monitoring of tumors.     

Impedimetric immunosensor using avidin-biotin for antibody immobilization

A newly developed electrochemical method--Elimination Voltammetry with Linear Scan (EVLS)--has been applied to the electrochemical study of nucleic acids (NAs) on a silver electrode. Using the linear combination of the currents measured at different scan rates, the EVLS is capable of eliminating one or two selected particular currents. It was shown that the elimination function conserving the reversible diffusion current and eliminating the charging and kinetic currents provides the significant increase of voltammetric signals of DNA. Due to the high sensitivity and resolution power, the EVLS can contribute to study behaviour of nucleic acids on the charged interface and can be applied to nucleic acid analyses and the development of DNA sensors.     

Quartz crystal microbalance genosensor for sequence specific detection of attomolar DNA targets

A mass sensitive quartz crystal microbalance (QCM) based genosensor has been developed using breast cancer 1 (BRCA1) gene as a model gene. We modified the traditional sandwich assay by conjugating reporter probe DNA (DNA-r) with an assembly of gold nanoparticles leading to an increased mass on the surface, which enhanced the sensitivity to few orders of magnitude. The unique cleavage function of endonuclease is used for achieving the selectivity to complementary DNA over mismatched DNA. With this combination, the sensor exhibited excellent sensitivity with a detection limit of 10 aM BRCA1 gene and it showed good selectivity for even single base mismatch DNA targets. This ultrasensitive and cost-effective DNA detection protocol can be extended to the direct analysis of any non-amplified genomic DNA.     

A new genosensor for meningococcal meningitis diagnosis using biological samples

In this work, a new electrochemical biosensor for DNA detection of bacterial meningitis is proposed. The system is based on specific DNA fragments from the Neisseria meningitidis genome as a probe incorporated on graphite electrodes modified with poly(4-aminophenol). Detection of a complementary oligonucleotide sequence, a specific 710-base pair amplicon, and the genomic DNA of bacteria was carried out by differential pulse voltammetry, using ethidium bromide as an electroactive indicator of hybridization. The complementary oligonucleotide and the genomic DNA of Neisseria meningitidis were quantified by the genosensor, showing detection limits of 0.6 ng μL^?1 and about 6 ng μL^?1, respectively. Morphological differences were observed between hybridized and unhybridized surfaces by atomic force microscopy. The biosensor showed high selectivity, discriminating non-specific targets, and high stability retaining over 98% of its original activity after 120 days of storage. The bioelectrode was effective in discriminating the genomic DNA in samples with human serum without significant interference, proving to be an interesting platform for meningococcal meningitis diagnosis.     

Impedimetric immunosensor for human serum albumin detection on a direct aldehyde-functionalized silicon nitride surface

In this work we report the fabrication and characterization of a label-free impedimetric immunosensor based on a silicon nitride (Si₃N₄) surface for the specific detection of human serum albumin (HSA) proteins. Silicon nitride provides several advantages compared with other materials commonly used, such as gold, and in particular in solid-state physics for electronic-based biosensors. However, few Si₃N₄-based biosensors have been developed; the lack of an efficient and direct protocol for the integration of biological elements with silicon-based substrates is still one of its the main drawbacks. Here, we use a direct functionalization method for the direct covalent binding of monoclonal anti-HSA antibodies on an aldehyde-functionalized Si-p/SiO₂/Si₃N₄ structure. This methodology, in contrast with most of the protocols reported in literature, requires less chemical reagents, it is less time-consuming and it does not need any chemical activation. The detection capability of the immunosensor was tested by performing non-faradaic electrochemical impedance spectroscopy (EIS) measurements for the specific detection of HSA proteins. Protein concentrations within the linear range of 10⁻¹³–10⁻⁷ M were detected, showing a sensitivity of 0.128 Ω μM⁻¹ and a limit of detection of 10⁻¹⁴ M. The specificity of the sensor was also addressed by studying the interferences with a similar protein, bovine serum albumin. The results obtained show that the antibodies were efficiently immobilized and the proteins detected specifically, thus, establishing the basis and the potential applicability of the developed silicon nitride-based immunosensor for the detection of proteins in real and more complex samples.     

Comparative proteomic profiling of mammalian cell lysates using phosphopeptide microarrays

A library of 176 human phosphotyrosine-containing peptides was used to establish cell lysate binding profiles in a two colour microarray format. The resulting hits led to the pull-down and identification of biomarkers associated with cancer states.     

Electrochemical genosensor for the detection of interaction between methylene blue and DNA

Described here are the chronocoulometric and voltammetric parameters for methylene blue [3,7-bis(dimethylamino)phenothiazin-5-ium chloride, MB] on binding to DNA at carbon paste electrode (CPE) surface. MB, which interacts with the immobilized calf thymus DNA was detected by using single stranded DNA modified CPE (ssDNA modified CPE), bare CPE and double stranded DNA modified CPE (dsDNA modified CPE) in combination with chronocoulometry and differential pulse voltammetry (DPV) techniques. The effect of ionic strength to the behavior of MB with dsDNA and ssDNA was also studied by means of voltammetry. These results demonstrated that MB could be used as an effective electroactive hybridization indicator for DNA biosensors. Performance characteristics of the sensor are described, along with future prospects.     

Impedimetric detection of trinitrotoluene by using a glassy carbon electrode modified with a gold nanoparticle@fullerene composite and an aptamer-imprinted polydopamine

Microchimica Acta - This study introduces a method based on a combination of aptasensing and molecular imprinting for highly sensitive detection of 2,4,6-trinitrotoluene (TNT). An amino-aptamer...     

Electrochemical genosensor array for the simultaneous detection of multiple high-risk human papillomavirus sequences in clinical samples

An electrochemical genosensor array for the simultaneous detection of three high-risk human papillomavirus (HPV) DNA sequences, HPV16, 18 and 45, exhibiting high sensitivity and selectivity is presented. The electrodes of a 4 × 4 array were modified via co-immobilization of a 1:100 (mol/mol) mixture of a thiolated probe and an oligoethyleneglycol-terminated bipodal thiol. Detection of synthetic and PCR products was carried out in a sandwich type format, with the target hybridized between a surface immobilized probe and a horseradish peroxidase-labelled secondary reporter probe. The detection limits obtained in the detection of each individual target were in the pM range, allowing the application of this sensor for the detection of samples obtained from PCR amplification of cervical scrape samples. The results obtained exhibited an excellent correlation with the HPV genotyping carried out within a hospital laboratory. Multiplexing and cross-reactivity studies demonstrated high selectivity over potential interfering sequences, facilitating application of the developed platform for the high-throughput screening of multiple high-risk DNA sequences.     

Picomolar detection of mercury (II) using a three-dimensional porous graphene/polypyrrole composite electrode

A polypyrrole (PPy)-functionalized three-dimensional (3D) porous electrode of electrochemically reduced graphene oxide (ErGO) has been prepared by electrochemical deposition. This PPy-modified 3D-ErGO electrode was used for the electrochemical detection of Hg²⁺ ions, and it exhibited high sensitivity and selectivity. Furthermore, the limit of detection (LOD) was measured to be as low as 0.03 nM (30 ppt), and this value is much lower than the guideline value of 2 ppb for drinking water given by the World Health Organization.     

A robust small-molecule microarray platform for screening cell lysates

Herein we report the expanded functional group compatibility of small-molecule microarrays to include immobilization of primary alcohols, secondary alcohols, phenols, carboxylic acids, hydroxamic acids, thiols, and amines on a single slide surface. Small-molecule "diversity microarrays" containing nearly 10,000 known bioactive small molecules, natural products, and small molecules originating from several diversity-oriented syntheses were produced by using an isocyanate-mediated covalent capture strategy. Selected printed bioactive compounds were detected with antibodies against compounds of interest. The new surface of the diversity microarrays is highly compatible with approaches involving cellular lysates. This feature has enabled a robust, optimized screening methodology using cellular lysates, allowing the detection of specific interactions with a broad range of binding affinity by using epitope-tagged or chimeric fluorescent proteins without prior purification. We believe that this expanded research capability has considerable promise in biology and medicine.     

Detection of telomerase activity in high concentration of cell lysates using primer-modified gold nanoparticles

Although the telomeric repeat amplification protocol (TRAP) has served as a powerful assay for detecting telomerase activity, its use has been significantly limited when performed directly in complex, interferant-laced samples. In this work, we report a modification of the TRAP assay that allows the detection of high-fidelity amplification of telomerase products directly from concentrated cell lysates. Briefly, we covalently attached 12 nm gold nanoparticles (AuNPs) to the telomere strand (TS) primer, which is used as a substrate for telomerase elongation. These TS-modified AuNPs significantly reduce polymerase chain reaction (PCR) artifacts (such as primer dimers) and improve the yield of amplified telomerase products relative to the traditional TRAP assay when amplification is performed in concentrated cell lysates. Specifically, because the TS-modified AuNPs eliminate most of the primer-dimer artifacts normally visible at the same position as the shortest amplified telomerase PCR product apparent on agarose gels, the AuNP-modified TRAP assay exhibits excellent sensitivity. Consequently, we observed a 10-fold increase in sensitivity for cancer cells diluted 1000-fold with somatic cells. It thus appears that the use of AuNP-modified primers significantly improves the sensitivity and specificity of the traditional TRAP assay and may be an effective method by which PCR can be performed directly in concentrated cell lysates.     

Energy-storable dye-sensitized solar cell with a polypyrrole electrode

A three-electrode-type solar-rechargeable battery, energy-storable dye-sensitized solar cell (ES-DSSC), has been constructed by the hybridization of a typical Gratzel cell and a conducting polymer charge-storage electrode; efficient photo-charging can be accomplished by visible-light irradiation.     

Electrochemical genosensor for detection of human mammaglobin in polymerase chain reaction amplification products of breast cancer patients

Human mammaglobin (MG) has been found to be the most specific molecular marker for the hematogenous spread of breast cancer cells. In our study, an electrochemical impedance spectroscopic DNA biosensor was established for the detection of MG in breast cancer patients. The working conditions for the biosensor, such as immobilization time, rinse process, and hybridization process, were optimized. Under the optimal conditions, the charge transfer resistance of the proposed DNA biosensor shows excellent correlation with the amount of the complementary oligonucleotides in the range from 1.0?×?10^?9 to 2.0?×?10^?8?M. The detection limit is 5.0?×?10^?10?M. The proposed biosensor was used to detect the polymerase chain reaction amplification products of actual clinical breast cancer samples. The results were compared with that obtained by conventional gel electrophoresis. The results indicate that the electrochemical impedance spectroscopic assay is significantly sensitive and time-saving. The simple strategy described here is expected to be used in clinical application for early diagnosis of breast cancer.