Carbon nanotube/polysulfone screen-printed electrochemical immunosensor

The simple and efficient method for preparing sensitive carbon nanotube/polysulfone/RIgG immunocomposite is described. The membrane of the modified disposable screen-printed electrochemical immunosensor is based on phase inversion method. Carbon nanotube/polysulfone membrane acts both as reservoir of immunological material and transducer while offering high surface area, high toughness and mechanical flexibility. The comparison with graphite/polysulfone/RIgG immunosensors shows a much higher sensitivity for those prepared with carbon nanotubes coupled with polysulfone (PSf). The membrane was characterized by scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDX), laser profilometer and by atomic force microscopy (AFM). The purity of the materials was evaluated by thermogravimetric analysis (TGA). The roughness value is doubled when MWCNTs are used instead of graphite into the PSf membranes and the incorporation of antibodies enhances the dispersion of the carbon with the polymeric membrane reducing the roughness in all cases. This biosensor was based on the competitive assay between free and labelled anti-RIgG for the available binding sites of immobilized rabbit IgG (RIgG). The RIgG was incorporated into the polysulfone membrane by a phase inversion method. Horse radish peroxidase (HRP) enzyme was used as label and hydroquinone as mediator. The detection limit for competitive assay was determined to be 1.66 microg/ml. the linear range of anti-RIgG from 2 to 5 microg/ml and the C(50) was found at 3.56 microg/ml. The sensitivity is five times higher for MWCNT than for graphite electrodes, showing lower unspecific adsorption.     

Direct electron transfer of Horseradish peroxidase on porous structure of screen-printed electrode

Disposable hydrogen peroxide biosensor was developed based on the direct electron transfer of horseradish peroxidase (HRP) on porous screen-printed carbon electrodes. Conventional screen-printing process was manually performed to fabricate the planar carbon electrodes, which were endowed with porous surfaces especially after anodizing pretreatment. The cyclic voltammetry experiment indicated a pair of stable and well-defined redox peaks with a formal potential of -0.33 V. And the formal potential was pH-dependent, having a slope of -55.2 mV/pH which indicated one electron transfer. The heterogeneous electron transfer rate constant k(s) was estimated to be 13.28+/-4.80s(-1). Additionally, the sensitivity was 143.3 mAM(-1)cm(-2) and the linear range was from 5.98 to 35.36 microM. In conclusion, the present work achieved the direct electron transfer of HRP on screen-printed electrodes without any promoters. The porous structure of screen-printed carbon electrodes facilitated the direct electron transfer between the active sites of HRP and the electrodes due to large amounts of conductive sites available on the surface for contacting with enzyme molecules. Moreover, the proposed biosensor could be mass-produced at low price, promising for commercial application.     

Celiac disease detection using a transglutaminase electrochemical immunosensor fabricated on nanohybrid screen-printed carbon electrodes

Celiac disease is a gluten-induced autoimmune enteropathy characterized by the presence of tissue tranglutaminase (tTG) autoantibodies. A disposable electrochemical immunosensor (EI) for the detection of IgA and IgG type anti-tTG autoantibodies in real patient's samples is presented. Screen-printed carbon electrodes (SPCE) nanostructurized with carbon nanotubes and gold nanoparticles were used as the transducer surface. This transducer exhibits the excellent characteristics of carbon-metal nanoparticle hybrid conjugation and led to the amplification of the immunological interaction. The immunosensing strategy consisted of the immobilization of tTG on the nanostructured electrode surface followed by the electrochemical detection of the autoantibodies present in the samples using an alkaline phosphatase (AP) labelled anti-human IgA or IgG antibody. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with a commercial ELISA kit indicating that the electrochemical immunosensor is a trustful analytical screening tool.     

Rapid identification of viable Escherichia coli subspecies with an electrochemical screen-printed biosensor array

Rapid identification of Escherichia coli strains is an important diagnostic goal in applied medicine as well as the environmental and food sciences. This paper reports an electrochemical, screen-printed biosensor array, where selective recognition is accomplished using lectins that recognize and bind to cell-surface lipopolysaccharides and coulometric transduction exploits non-native external oxidants to monitor respiratory cycle activity in lectin-bound cells. Ten different lectins were separately immobilized onto porous membranes that feature activated surfaces (ImmunodyneABC). Modified membranes were exposed to untreated E. coli cultures for 30 min, rinsed, and layered over the individual screen-printed carbon electrodes of the sensor array. The membranes were were incubated 5 min in a reagent solution that contained the oxidants menadione and ferricyanide as well as the respiratory substrates succinate and formate. Electrochemical oxidation of ferrocyanide for 2 min provided chronocoulometric data related to the quantities of bound cells. These screen-printed sensor arrays were used in conjunction with factor analysis for the rapid identification of four E. coli subspecies (E. coli B, E. coli Neotype, E. coli JM105 and E. coli HB101). Systematic examination of lectin-binding patterns showed that these four E. coli subspecies are readily distinguished using only five essential lectins.     

A disposable electrochemical immunosensor based on carbon screen-printed electrodes for the detection of prostate specific antigen

A novel screen-printed electrode (SPEs) on sheets of vegetable parchment was prepared. The obtained SPEs were stable, convenient, inexpensive and suitable for large-area screen-printing. With these SPEs, we explored the fabrication of a novel, disposable and highly sensitive electro-analytical immunosensor using graphene nanosheets (GS) and horseradish peroxidase (HRP)-labeled signal antibody functionalized with gold nanoparticles (HRP-Ab(2)/Au NPs). GS was used to increase the conductivity and stability of this immunosensor due to its fast electron transportation and good biocompatibility. Au NPs could not only provide a large surface area for the immobilization of HRP-Ab(2) but also enhance the electroreduction between HRP and H(2)O(2) to amplify the electrochemical signal on the sandwich immuno-complexes modified SPEs. The proposed SPEs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and electrochemical methods involving cyclic voltammetry (CV), and electrochemical impedence method. Using prostate specific antigen (PSA) as a model analyte, this immunosensor showed a wide linear range over 6 orders of magnitude with the minimum value down to 2pgmL(-1). In addition, this immunosensor could avoid the need of deoxygenation for the electrochemical immunoassay. Thus, it provided a promising potential in clinical applications.     

An electrochemical immunosensor for aflatoxin M1 determination in milk using screen-printed electrodes

The production and assembling of disposable electrochemical AFM1 immunosensors, which can combine the high selectivity of immunoanalysis with the ease of the electrochemical probes, has been carried out. Firstly immunoassay parameters such as amounts of antibody and labelled antigen, buffer and pH, length of time and temperature of each steps (precoating, coating, binding and competition steps) were evaluated and optimised in order to set up a spectrophotometric enzyme-linked immunosorbent assay (ELISA) procedure. This assay exhibited a working range between 30 and 160 ppt in a direct competitive format. Then electrochemical immunosensors were fabricated by immobilising the antibodies directly on the surface of screen-printed electrodes (SPEs), and allowing the competition to occur between free AFM1 and that conjugated with peroxidase (HRP) enzyme. The electrochemical technique chosen was the chronoamperometry, performed at -100 mV. Furthermore, studies of interference and matrix effects have been performed to evaluate the suitability of the developed immunosensors for the analysis of aflatoxin M1 directly in milk. Results have shown that using screen-printed electrodes aflatoxin M1 can be measured with a detection limit of 25 ppt and with a working range between 30 and 160 ppt. A comparison between the spectrophotometric and electrochemical procedure showed that a better detection limit and shorter analysis time could be achieved using electrochemical detection.     

Direct electron transfer between cytochrome P450scc and gold nanoparticles on screen-printed rhodium-graphite electrodes

This paper is concerned with an investigation of electron transfer between cytochrome P450scc (CYP11A1) and gold nanoparticles immobilised on rhodium-graphite electrodes. Thin films of gold nanoparticles were deposited onto the rhodium-graphite electrodes by drop casting. Cytochrome P450scc was deposited onto both gold nanoparticle modified and bare rhodium-graphite electrodes. Cyclic voltammetry indicated enhanced activity of the enzyme at the gold nanoparticle modified surface. The role of the nanoparticles in mediating electron transfer to the cytochrome P450scc was verified using ac impedance spectroscopy. Equivalent circuit analysis of the impedance spectra was performed and the values of the individual components estimated. On addition of aliquots of cholesterol to the electrolyte bioelectrocatalytic reduction currents were obtained. The sensitivity of the nanoparticle modified biosensor to cholesterol was 0.13 microA microM-1 in a detection range between 10 and 70 microM of cholesterol. This confirms that gold nanoparticles enhance electron transfer to the P450scc when present on the rhodium-graphite electrodes.     

Identification of upper respiratory tract pathogens using electrochemical detection on an oligonucleotide microarray

Bacterial and viral upper respiratory infections (URI) produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD) on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae) and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluenza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.     

Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation

There are many sources of systematic variation in cDNA microarray experiments which affect the measured gene expression levels (e.g. differences in labeling efficiency between the two fluorescent dyes). The term normalization refers to the process of removing such variation. A constant adjustment is often used to force the distribution of the intensity log ratios to have a median of zero for each slide. However, such global normalization approaches are not adequate in situations where dye biases can depend on spot overall intensity and/or spatial location within the array. This article proposes normalization methods that are based on robust local regression and account for intensity and spatial dependence in dye biases for different types of cDNA microarray experiments. The selection of appropriate controls for normalization is discussed and a novel set of controls (microarray sample pool, MSP) is introduced to aid in intensity-dependent normalization. Lastly, to allow for comparisons of expression levels across slides, a robust method based on maximum likelihood estimation is proposed to adjust for scale differences among slides.     

An electrochemical biosensor for 3-hydroxybutyrate detection based on screen-printed electrode modified by coenzyme functionalized carbon nanotubes

3-Hydroxybutyrate, one of the main blood ketone bodies, has been considered as a critical indicator for diagnosis of diabetic ketoacidosis. Biosensors designed for detection of 3-hydroxybutyrate with advantages of precision, easiness and speedy performance have attracted increasing attention. This study attempted to develop a 3-hydroxybutyrate dehydrogenase-based biosensor in which single-walled carbon nanotubes (SWCNT) was used in order to immobilize the cofactor, NAD⁺, on the surface of screen-printed electrode. The formation of NAD⁺–SWCNT conjugates was assessed by electrochemistry and electron microscopy. Cyclic voltammetry was used to analyze the performance of this biosensor electrochemically. The considerable shelf life and reliability of the proposed biosensor to analyze real sample was confirmed by this method. The reduction in the over potential of electrochemical oxidation of NADH to ?0.15 V can be mentioned as a prominent feature of this biosensor. This biosensor can detect 3-hydroxybutyrate in the linear range of 0.01–0.1 mM with the low detection limit of 0.009 mM. Simultaneous application of screen-printed electrode and SWCNT has made the biosensor distinguished which can open new prospects for detection of other clinically significant metabolites.     

Direct selection of human genomic loci by microarray hybridization

We applied high-density microarrays to the enrichment of specific sequences from the human genome for high-throughput sequencing. After capture of 6,726 approximately 500-base 'exon' segments, and of 'locus-specific' regions ranging in size from 200 kb to 5 Mb, followed by sequencing on a 454 Life Sciences FLX sequencer, most sequence reads represented selection targets. These direct selection methods supersede multiplex PCR for the large-scale analysis of genomic regions.     

Development of a screen-printed carbon electrochemical immunosensor for picomolar concentrations of estradiol in human serum extracts

Investigations into the development of a prototype electrochemical immunosensor for estradiol (E(2)) are described. After optimising reagent loadings in a 96-well enzyme-linked immunosorbent assay (ELISA), antibodies (rabbit anti-mouse IgG and monoclonal mouse anti-E(2)) were immobilised by passive adsorption onto the surface of screen-printed carbon electrodes (SPCEs). A competitive immunoassay was then performed using an alkaline-phosphatase (ALP)-labelled E(2) conjugate. Calibration plots for E(2) buffer standards, performed colorimetrically on the SPCEs using a para-nitrophenyl phosphate substrate solution, were in good agreement with ELISA calibration plots. Electrochemical measurements were then performed using differential pulse voltammetry (DPV) following the production of 1-naphthol from 1-naphthyl phosphate. The calibration plot of DPV peak current versus E(2) concentration showed a measurable range of 25-500 pg/ml with a detection limit of 50 pg/ml. A coefficient of variation of between 13.0 and 15.6% was obtained for repeat measurements. The immunosensor was applied to the determination of E(2) in spiked serum, following an extraction step with diethyl ether. A mean recovery for the method of 102.5% was obtained with a CV of 19.1%. The options available for further development of the sensor regarding precision, limit of detection and direct sample analysis are discussed.     

Direct electrochemical determination of glucose oxidase in biological samples

An electrochemical method for the quantitation of glucose oxidase in murine plasma and tissues has been developed. Instead of oxygen, this method uses benzoquinone as an artificial cosubstrate of glucose oxidase. The quantitative detection of the enzymatically produced hydroquinone by controlled-potential amperometry allows measurement of glucose oxidase concentrations in biological samples. The use of an internal standard corrects for all possible interfering effects. We demonstrated a 10-fold increase in sensitivity, as well as the ability to work in turbid media, in comparison to spectrophotometric methods.     

Direct detection of Staphylococcus aureus mRNA using a flow through microarray

The direct detection of mRNAs from bacterial cultures on a DNA array without amplification and labelling would greatly extend the range of applications suitable for microarray analysis. Here we describe the direct detection of 23S rRNA and seven mRNA species from total Staphylococcus aureus RNA prepared using commercially available RNA purification columns followed by fluorescent detection on a flow through microarray. RNA hybridisation was detected using paired secondary labelled probes directly 5' and 3' to immobilised 60 mers. In this way, we were able to detect the effect of 30-min exposure to antimicrobials on mRNA levels within 3 h after column purification of total RNA without the need for enzymatic manipulation. Specifically the expression of mecA was confirmed in a highly resistant strain and induction of katA and ile-tRNA synthetase genes after exposure to mupirocin could be detected.     

Glycopeptides of immunoglobulins: Investigations on IgA myeloma globulins

The oligosaccharide units of a type K and a type L IgA immunoglobulin have been examined. The two proteins differed in their content of 6-deoxy-l-galactose and N-acetylneuraminic acid, and in the d-mannose/d-galactose ratio. With glycopeptides prepared from the type K protein, specific glycosidases liberated the N-acetylneuraminic acid and 7-8 residues of 2-acetamido-2-deoxy-d-glucose, and mild acid hydrolysis released most of the 6-deoxy-l-galactose. The type K immunoglobulin appeared to contain 3 oligosaccharide units, whereas the type L protein probably contained 3 or more units.     

Graphitized macroporous carbon microarray with hierarchical mesopores as host for the fabrication of electrochemical biosensor

A novel graphitized ordered macroporous carbon (GMC, pore size 380 nm) with hierarchical mesopores (2–30 nm) and high graphitization degree was prepared by nickel-catalyzed graphitization of polystyrene arrays. The obtained GMC possessed high specific surface area, large pore volume, and good electrical conductivity, which was explored for the enzyme entrapment and biosensor fabrication by a facile method. With advantages of novel nanostructure and good electrical conductivity, direct electrochemistry of hemoglobin (a model protein) was observed on the GMC-based biocomposite with a formal potential of −0.36 V (vs. Ag/AgCl) and an apparent heterogeneous electron transfer rate constant (k_s) of 1.2 s⁻¹ in pH 7.0 buffer. Comparative studies revealed that GMC offered significant advantages over carbon nanotubes (CNTs) in facilitating direct electron transfer of entrapped Hb. The fabricated biosensor exhibited good sensitivity (101.6 mA cm⁻² M⁻¹) and reproducibility, wide linear range (1–267 μM), low detection limit (0.1 μM), and good long-term stability for H₂O₂ detection. GMC proved to be a promising matrix for enzyme entrapment and biosensor fabrication, and may find wide potential applications in biomedical detection and environmental analyses.     

Direct immobilization of antibodies on a new polymer film for fabricating an electrochemical impedance immunosensor

A new polymer bearing aldehyde groups was designed and synthesized by grafting 4-pyridinecarboxaldehyde onto poly(epichlorohydrin). Antibodies can be directly immobilized on the surface of the polymer film through the covalent bonding of aldehyde groups of the film with amino groups of antibodies. In this study, human immunoglobulin G (IgG) was used as a model analyte for the fabrication of an electrochemical impedance immunosensor. Using the proposed immunosensor, IgG in the range from 0.1 to 80 ng ml⁻¹ was detected with a detection limit of 0.07 ng ml⁻¹ (signal/noise [S/N] = 3). In addition, the electrochemical impedance immunosensor displays good stability and reproducibility.     

Proteomics: addressing the challenges of multiple myeloma

Multiple myeloma (MM) is a malignancy of terminally differentiated B-lymphocytes that accounts for ~13% of all hematologic cancers. Despite a wealth of knowledge describing the molecular biology of MM as well as significant advances in therapeutics, this disease remains incurable. Since proteins govern the cellular structure and biological function, a wide selection of proteomic approaches holds great promise for increasing our understanding of this disease, such as by investigating the dynamic nature of protein expression, cellular and subcellular distribution, post-translational modifications, and interactions at both the cellular and subcellular levels. The aims of this review are to introduce the available and emerging proteomic technologies that have potential applications in the study of MM and to highlight the current status of proteomic studies of MM. To date, although there have been a limited number of proteomic studies in MM, those performed have provided valuable information with regard to MM diagnosis and therapy. The potential future application of proteomic technologies is expected to provide new avenues in MM diagnostics, individualized therapy design and therapy response surveillance for the clinician.     

Generalization of DNA microarray dispersion properties: microarray equivalent of t-distribution

Background

DNA microarrays are a powerful technology that can provide a wealth of gene expression data for disease studies, drug development, and a wide scope of other investigations. Because of the large volume and inherent variability of DNA microarray data, many new statistical methods have been developed for evaluating the significance of the observed differences in gene expression. However, until now little attention has been given to the characterization of dispersion of DNA microarray data.

Results

Here we examine the expression data obtained from 682 Affymetrix GeneChips^® with 22 different types and we demonstrate that the Gaussian (normal) frequency distribution is characteristic for the variability of gene expression values. However, typically 5 to 15% of the samples deviate from normality. Furthermore, it is shown that the frequency distributions of the difference of expression in subsets of ordered, consecutive pairs of genes (consecutive samples) in pair-wise comparisons of replicate experiments are also normal. We describe a consecutive sampling method, which is employed to calculate the characteristic function approximating standard deviation and show that the standard deviation derived from the consecutive samples is equivalent to the standard deviation obtained from individual genes. Finally, we determine the boundaries of probability intervals and demonstrate that the coefficients defining the intervals are independent of sample characteristics, variability of data, laboratory conditions and type of chips. These coefficients are very closely correlated with Student's t-distribution.

Conclusion

In this study we ascertained that the non-systematic variations possess Gaussian distribution, determined the probability intervals and demonstrated that the K _α coefficients defining these intervals are invariant; these coefficients offer a convenient universal measure of dispersion of data. The fact that the K _α distributions are so close to t-distribution and independent of conditions and type of arrays suggests that the quantitative data provided by Affymetrix technology give "true" representation of physical processes, involved in measurement of RNA abundance.

Reviewers

This article was reviewed by Yoav Gilad (nominated by Doron Lancet), Sach Mukherjee (nominated by Sandrine Dudoit) and Amir Niknejad and Shmuel Friedland (nominated by Neil Smalheiser).