Search

Despite the excellent diagnostic applications of the current conventional immunoassay methods such as ELISA, immunostaining and Western blot for FAM134B detection, they are laborious, expensive and required a long turnaround time. Here, we report an electrochemical approach for rapid, sensitive, and specific detection of FAM134B protein in biological (colon cancer cell extracts) and clinical (serum) samples. The approach utilises a differential pulse voltammetry (DPV) in the presence of the [Fe(CN) 6 ] 3-/4- redox system to quantify the FAM134B protein in a two-step strategy that involves (i) initial attachment of FAM134B antibody on the surface of extravidin-modified screen-printed carbon electrode, and (ii) subsequent detection of FAM134B protein present in the biological/clinical samples. The assay system was able to detect FAM134B protein at a concentration down to 10 pg μL -1 in phosphate buffered saline (pH 7.4) with a good inter-assay reproducibility (% RSD = <8.64, n = 3). We found excellent sensitivity and specificity for the analysis of FAM134B protein in a panel of colon cancer cell lines and serum samples. Finally, the assay was further validated with ELISA method. We believe that our assay could potentially lead a low-cost alternative to conventional immunological assays for target antigens analysis in point-of-care applications.

Rapid progress in disease biomarker discovery has increased the need for robust detection technologies. In the past several years, the designs of many immunoaffinity reagents have focused on lowering costs and improving specificity while also promoting stability. Antibody fragments (scFvs) have long been displayed on the surface of yeast and phage libraries for selection; however, the stable production of such fragments presents challenges that hamper their widespread use in diagnostics. Membrane and cell wall proteins similarly suffer from stability problems when solubilized from their native environment. Recently, cell envelope compositions that maintain membrane proteins in native or native-like lipid environment to improve their stability have been developed. This cell envelope composition approach has now been adapted toward stabilizing antibody fragments by retaining their native cell wall environment. A new class of immunoaffinity reagents has been developed that maintains antibody fragment attachment to yeast cell wall. Herein, we review recent strategies that incorporate cell wall fragments with functional scFvs, which are designed for easy production while maintaining specificity and stability when in use with simple detection platforms. These cell wall based antibody fragments are globular in structure, and heterogeneous in size, with fragments ranging from tens to hundreds of nanometers in size. These fragments appear to retain activity once immobilized onto biosensor surfaces for the specific and sensitive detection of pathogen antigens. They can be quickly and economically generated from a yeast display library and stored lyophilized, at room temperature, for up to a year with little effect on stability. This new format of scFvs provides stability, in a simple and low-cost manner toward the use of scFvs in biosensor applications. The production and "panning" of such antibody cell wall composites are also extremely facile, enabling the rapid adoption of stable and inexpensive affinity reagents for emerging infectious threats.

The synthesis of a novel family of cyano-bridged trimetallic coordination polymers (CPs) with various compositions and shapes has been reported by changing the compositional ratios of Fe, Co, and Ni species in the reaction system. In order to efficiently control the nucleation rate and the crystal growth, trisodium citrate dihydrate plays an important role as a chelating agent. After the obtained cyano-bridged trimetallic CPs undergo thermal treatment in air at three different temperatures (250, 350, and 450 °C), nanoporous spinel metal oxides are successfully obtained. Interestingly, the obtained nanoporous metal oxides are composed of small crstalline grains, and the grains are oriented in the same direction, realizing pseudo-single crystals with nanopores. The resultant nanoporous spinel oxides feature interesting magnetic properties. Cyano-bridged multimetallic CPs with various sizes and shapes can provide a pathway toward functional nanoporous metal oxides that are not attainable from simple cyano-bridged CPs containing single metal ions., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tumor-derived exosomes possess significant clinical relevance due to their unique composition of genetic and protein material that is representative of the parent tumor. Specific isolation as well as identification of proportions of these clinically relevant exosomes (CREs) from biological samples could help to better understand their clinical significance as cancer biomarkers. Herein, we present a simple approach for quantification of the proportion of CREs within the bulk exosome population isolated from patient serum. This proportion of CREs can potentially inform on the disease stage and enable non-invasive monitoring of inter-individual variations in tumor-receptor expression levels. Our approach utilises a Surface Plasmon Resonance (SPR) platform to quantify the proportion of CREs in a two-step strategy that involves (i) initial isolation of bulk exosome population using tetraspanin biomarkers (i.e., CD9, CD63), and (ii) subsequent detection of CREs within the captured bulk exosomes using tumor-specific markers (e.g., human epidermal growth factor receptor 2 (HER2)). We demonstrate the isolation of bulk exosome population and detection of as low as 10% HER2(+) exosomes from samples containing designated proportions of HER2(+) BT474 and HER2(-) MDA-MB-231 cell derived exosomes. We also demonstrate the successful isolation of exosomes from a small cohort of breast cancer patient samples and identified that approximately 14-35% of their bulk population express HER2.

A crucial issue in present-day prostate cancer (PCa) detection is the lack of specific biomarkers for accurately distinguishing between benign and malignant cancer forms. This is causing a high degree of overdiagnosis and overtreatment of otherwise clinically insignificant cases. As around half of all malignant PCa cases display a detectable gene fusion mutation between the TMPRSS2 promoter sequence and the ERG coding sequence (TMPRSS2:ERG) in urine, noninvasive screening of TMPRSS2:ERG mRNA in patient urine samples could improve the specificity of current PCa diagnosis. However, current gene fusion detection methodologies are largely dependent on RNA enzymatic amplification, which requires extensive sample manipulation, costly labels for detection, and is prone to bias/artifacts. Herein we introduce the first successful amplification-free electrochemical assay for direct detection of TMPRSS2:ERG mRNA in PCa urinary samples by selectively isolating and adsorbing TMPRSS2:ERG mRNA onto bare gold electrodes without requiring any surface modification. We demonstrated excellent limit-of-detection (10 cells) and specificity using PCa cell line models, and showcased clinical utility by accurately detecting TMPRSS2:ERG in a collection of 17 urinary samples obtained from PCa patients. Furthermore, these results were validated with the current gold standard reverse transcription (RT)-PCR approach with 100% concordance.

Mutation of FAM134B (Family with Sequence Similarity 134, Member B) leading to loss of function of its encoded Golgi protein and has been reported induce apoptosis in neurological disorders. FAM134B mutation is still unexplored in cancer. Herein, we studied the DNA copy number variation and novel mutation sites of FAM134B in a large cohort of freshly collected oesophageal squamous cell carcinoma (ESCC) tissue samples. In ESCC tissues, 37% (38/102) showed increased FAM134B DNA copies whereas 35% (36/102) showed loss of FAM134B copies relative to matched non-cancer tissues. Novel mutations were detected in exons 4, 5, 7, 9 as well as introns 2, 4-8 of FAM134B via HRM (High-Resolution Melt) and Sanger sequencing analysis. Overall, thirty-seven FAM134B mutations were noted in which most (31/37) mutations were homozygous. FAM134B mutations were detected in all the cases with metastatic ESCC in the lymph node tested and in 14% (8/57) of the primary ESCC. Genetic alteration of FAM134B is a frequent event in the progression of ESCCs. These findings imply that mutation might be the major driving source of FAM134B genetic modulation in ESCCs.

Whilst recent advances in nanotechnology have yielded many new biosensing capabilities, innovative biological attachment and detection modalities remain relatively underdeveloped. Bi-specific antibodies (bsAbs)--which exhibit binding capability for two separate targets--offer an inherent advantage over conventional antibody reagents by significantly simplifying sensor surface preparation. Herein, we report the deployment of bsAbs for simultaneous attachment to a polymer-coated transducer and label-free, electrochemical (EC) detection of target antigens.

We report a new method for the electrochemical detection of glycosylation on proteins, which relies on lectin-protein interaction on a bare gold electrode. The target protein isolated by immunoaffinity is directly adsorbed onto a gold surface and its glycosylation status is retrieved by subsequent addition of specific lectins. The adsorption and subsequent recognition process is monitored electrochemically in the presence of [Fe(CN)6](3-/4-) redox system. By decoupling target protein capture from glycosylation read-out steps, this approach circumvents unwanted antibody-lectin crosstalk while enabling specific glycosylation detection of a glycoprotein in serum-spiked samples in less than 1 h.

Current amplification-based microRNA (miRNA) detection approaches are limited by the small sizes of miRNAs as well as amplification bias/artifacts. Herein, we report on an amplification-free miRNA assay based on elevated affinity interaction between polyadenylated miRNA and bare gold electrode. The poly(A) extension on the 3' ends of magnetically isolated miRNA targets facilitated high adsorption efficiency onto gold electrode surfaces for electrochemical detection without any cumbersome electrode surface functionalization procedures. The assay showed excellent detection sensitivity (10 fM) and specificity and was demonstrated for quantitative miR-107 detection in human cancer cell lines and clinical urine samples. We believe our assay could be useful as an amplification-free alternative for miRNA detection.

With new systemic therapies becoming available for metastatic melanoma such as BRAF and PD-1 inhibitors, there is an increasing demand for methods to assist with treatment selection and response monitoring. Quantification and characterisation of circulating melanoma cells (CMCs) has been regarded as an excellent non-invasive candidate but a sensitive and efficient tool to do these is lacking. Herein we demonstrate a microfluidic approach for melanoma cell capture and subsequent on-chip evaluation of BRAF mutation status. Our approach utilizes a recently discovered alternating current electrohydrodynamic (AC-EHD)-induced surface shear forces, referred to as nanoshearing. A key feature of nanoshearing is the ability to agitate fluid to encourage contact with surface-bound antibody for the cell capture whilst removing nonspecific cells from the surface. By adjusting the AC-EHD force to match the binding affinity of antibodies against the melanoma-associated chondroitin sulphate proteoglycan (MCSP), a commonly expressed melanoma antigen, this platform achieved an average recovery of 84.7% from biological samples. Subsequent staining with anti-BRAF(V600E) specific antibody enabled on-chip evaluation of BRAF(V600E) mutation status in melanoma cells. We believe that the ability of nanoshearing-based capture to enumerate melanoma cells and subsequent on-chip characterisation has the potential as a rapid screening tool while making treatment decisions.

Electrohydrodynamics (EHD) deals with the fluid motion induced by an electric field. This phenomenon originally developed in physical science, and engineering is currently experiencing a renaissance in microfluidics. Investigations by Taylor on Gilbert's theory proposed in 1600 have evolved to include multiple contributions including the promising effects arising from electric field interactions with cells and particles to influence their behaviour on electrode surfaces. Theoretical modelling of electric fields in microsystems and the ability to determine shear forces have certainly reached an advanced state. The ability to deftly manipulate microscopic fluid flow in bulk fluid and at solid/liquid interfaces has enabled the controlled assembly, coagulation, or removal of microstructures, nanostructures, cells, and molecules on surfaces. Furthermore, the ability of electrohydrodynamics to generate fluid flow using surface shear forces generated within nanometers from the surface and their application in bioassays has led to recent advancements in biomolecule, vesicle and cellular detection across different length scales. With the integration of Alternating Current Electrohydrodynamics (AC-EHD) in cellular and molecular assays proving to be highly fruitful, challenges still remain with respect to understanding the discrepancies between each of the associated ac-induced fluid flow phenomena, extending their utility towards clinical diagnostic development, and utilising them in tandem as a standard tool for disease monitoring. In this regard, this article will review the history of electrohydrodynamics, followed by some of the recent developments in the field including a new dimension of electrohydrodynamics that deals with the utilization of surface shear forces for the manipulation of biological cells or molecules on electrode surfaces. Recent advances and challenges in the use of electrohydrodynamic forces such as dielectrophoresis and ac electrosmosis for the detection of biological analytes are also reviewed. Additionally, the fundamental mechanisms of fluid flow using electrohydrodynamics forces, which are still evolving, are reviewed. Challenges and future directions are discussed from the perspective of both fundamental understanding and potential applications of these nanoscaled shear forces in diagnostics.

New high-performance detection technologies and more robust protein capture agents can be combined to both rapidly and specifically capture and detect protein biomarkers associated with disease in complex biological samples. Here we demonstrate the use of recently developed recombinant affinity reagents, namely nanoyeast-scFv, in combination with alternating current electrohydrodynamic (ac-EHD)-induced shear forces, to enhance capture performance during protein biomarker analysis. The use of ac-EHD significantly improves fluid transport across the capture domain, resulting in enhanced sensor-target interaction and simultaneous displacement of nonspecific molecules from the electrode surface. We demonstrate this simple proof-of-concept approach for the capture and detection of Entamoeba histolytica antigens from disinfected stool, within a span of 5 min using an ac-EHD microfluidic device. Under an ac-EHD field, antigens were captured on a nanoyeast-scFv immobilized device and subsequently detected using a quantum dot conjugated antibody. This immunosensor specifically detected antigen in disinfected stool with low background noise at concentrations down to 58.8 fM with an interassay reproducibility (%RSD of n = 3) < 17.2%, and in buffer down to 5.88 fM with an interassay reproducibility (% RSD, n = 3) of 8.4%. Furthermore, antigen detection using this immunosensor was 10 times more sensitive than previously obtained with the same nanoyeast-scFv reagents in a microfluidic device employing surface-enhanced Raman scattering (SERS) detection in buffer and at least 200 times more sensitive than methods using screen printed gold electrodes in disinfected stool. We predict this rapid and sensitive approach using these stable affinity reagents may offer a new methodology to detect protein disease biomarkers from biological matrices.

A rapid and simple approach is presented to address two critical issues of surface-enhanced Raman scattering (SERS)-based immunoassay such as removal/avoiding nonspecific adsorption and reducing assay time. The approach demonstrated involves rationally designed fluorophore-integrated gold/silver nanoshells as SERS nanotags and utilizes alternative current electrohydrodynamic (ac-EHD)-induced nanoscaled surface shear forces to enhance the capture kinetics. The assay performance was validated in comparison with hydrodynamic flow and conventional immunoassay-based devices. These nanoscaled physical forces acting within nanometer distances from the electrode surface enabled rapid (40 min), sensitive (10 fg/mL), and highly specific detection of human epidermal growth factor receptor 2 in breast cancer patient samples. We believe this approach presents potential for the development of rapid and sensitive SERS immunoassays for routine clinical diagnosis.

Microfluidic flow based multiplexed devices have gained significant promise in detecting biomarkers in complex biological samples. However, to fully exploit their use in bioanalysis, issues such as (i) low sensitivity and (ii) high levels of nonspecific adsorption of non-target species have to be overcome. Herein, we describe a new multiplexed device for the sensitive detection of multiple protein biomarkers in serum by using an alternating current (ac) electrohydrodynamics (ac-EHD) induced surface shear forces based phenomenon referred to as nanoshearing. The tunable nature (via manipulation of ac field) of these nanoshearing forces can alter the capture performance of the device (e.g., improved fluid transport enhances number of sensor-target collisions). This can also selectively displace weakly (nonspecifically) bound molecules from the electrode surface (i.e., fluid shear forces can be tuned to shear away nonspecific species present in biological samples). Using this approach, we achieved sensitive (100 fg mL(-1)) naked eye detection of multiple protein targets spiked in human serum and a 1000-fold enhancement in comparison to hydrodynamic flow based devices for biomarker detection. We believe that this approach could potentially represent a clinical diagnostic tool that can be integrated into resource-limited settings for sensitive detection of target biomarkers using naked eye.

Background: DNA methylation is a potential source of disease biomarkers. Typically, methylation levels are measured at individual cytosine/guanine (CpG) sites or over a short region of interest. However, regions of interest often show heterogeneous methylation comprising multiple patterns of methylation (epialleles) on individual DNA strands. Heterogeneous methylation is largely ignored because digital methods are required to deconvolute these usually complex patterns of epialleles. Currently, only single-molecule approaches, such as next generation sequencing (NGS), can provide detailed epiallele information. Because NGS is not yet feasible for routine practice, we developed a single-molecule-like approach, named for epiallele quantification (EpiQ)., Methods: EpiQ uses DNA ligases and the enhanced thermal instability of short (≤19 bases) mismatched DNA probes for the relative quantification of epialleles. The assay was developed using fluorescent detection on a gel and then adapted for electrochemical detection on a microfabricated device. NGS was used to validate the analytical accuracy of EpiQ., Results: In this proof of principle study, EpiQ detected with 90%-95% specificity each of the 8 possible epialleles for a 3-CpG cluster at the promoter region of the CDKN2B (p15) tumor suppressor gene. EpiQ successfully profiled heterogeneous methylation patterns in clinically derived samples, and the results were cross-validated with NGS., Conclusions: EpiQ is a potential alternative tool for characterizing heterogeneous methylation, thus facilitating its use as a biomarker. EpiQ was developed on a gel-based assay but can also easily be adapted for miniaturized chip-based platforms., (© 2014 American Association for Clinical Chemistry.)

Simple, sensitive and inexpensive regional DNA methylation detection methodologies are imperative for routine patient diagnostics. Herein, we describe eMethylsorb, an electrochemical assay for quantitative detection of regional DNA methylation on a single-use and cost-effective screen-printed gold electrode (SPE-Au) platform. The eMethylsorb approach is based on the inherent differential adsorption affinity of DNA bases to gold (i.e. adenine > cytosine ≥ guanine > thymine). Through bisulfite modification and asymmetric PCR of DNA, methylated and unmethylated DNA in the sample becomes guanine-enriched and adenine-enriched respectively. Under optimized conditions, adenine-enriched unmethylated DNA (higher affinity to gold) adsorbs more onto the SPE-Au surface than methylated DNA. Higher DNA adsorption causes stronger coulombic repulsion and hinders reduction of ferricyanide [Fe(CN)6](3-)ions on the SPE-Au surface to give a lower electrochemical response. Hence, the response level is directly proportional to the methylation level in the sample. The applicability of this methodology was tested by detecting the regional methylation status in a cluster of eight CpG sites within the engrailed (EN1) gene promoter of the MCF7 breast cancer cell line. A 10% methylation level sensitivity with good reproducibility (RSD = 5.8%, n = 3) was achieved rapidly in 10 min. Furthermore, eMethylsorb also has advantages over current methylation assays such as being inexpensive, rapid and does not require any electrode surface modification. We thus believe that the eMethylsorb assay could potentially be a rapid and accurate diagnostic assay for point-of-care DNA methylation analysis.

Aberrant protein glycosylation is associated with a range of pathological conditions including cancer and possesses diagnostic importance. Translation of glycoprotein biomarkers will be facilitated by the development of a rapid and sensitive analytical platform that simultaneously interrogates both the glycan and protein epitopes of glycoproteins in body fluids such as serum or saliva. To this end, we developed an electrochemical biosensor based on the immobilization of a lectin on the gold electrode surface to recognize/capture a target glycan epitope conjugated to glycoproteins, followed by detection of the protein epitope using a target protein-specific antibody. Electrochemical signals are generated by label-free voltammetric or impedimetric interrogation of a ferro/ferricyanide redox couple (e.g. [Fe(CN)6](3-/4-)) on the sensing surface, where the change in voltammetric current or interfacial electron transfer resistance was measured. The detection system was demonstrated using the model glycoprotein chicken ovalbumin with Sambucus nigra agglutinin type I (SNA lectin), and exhibits femtomolar sensitivity in the background of diluted human serum. The results obtained in this proof-of-concept study demonstrate the possibility of using electrochemical detection for developing cheap point-of-care diagnostics with high specificity and sensitivity for blood glycoprotein biomarkers.

Exosomes show promise as noninvasive biomarkers for cancer, but their effective capture and specific detection is a significant challenge. Herein, we report a multiplexed microfluidic device for highly specific capture and detection of multiple exosome targets using a tunable alternating current electrohydrodynamic (ac-EHD) methodology, referred to as nanoshearing. In our system, electrical body forces generated by ac-EHD act within nanometers of an electrode surface (i.e., within the electrical layer) to generate nanoscaled fluid flow that enhances the specificity of capture and also reduce nonspecific adsorption of weakly bound molecules from the electrode surface. This approach demonstrates the analysis of exosomes derived from cells expressing human epidermal growth factor receptor 2 (HER2) and prostate specific antigen (PSA), and is also capable of specifically isolating exosomes from breast cancer patient samples. The device also exhibited a 3-fold enhancement in detection sensitivity in comparison to hydrodynamic flow based assays (LOD 2760 exosomes/μL for ac-EHD vs LOD 8300 exosomes/μL for hydrodynamic flow; (n = 3)). We propose this approach can potentially have relevance as a simple and rapid quantification tool to analyze exosome targets in biological applications.

We report a simple method to remove nonspecifically adsorbed species from sensor surface and also improve the detection sensitivity of the sensor using tuneable alternating current (ac) electrohydrodynamics (ac-EHD) forces. These forces generated within few nanometers of an electrode surface (i.e., double layer) engender fluid flow within a serpentine channel containing a long array of the asymmetric electrode pairs, and can easily be tuned externally by changing the frequency and amplitude of the ac-EHD field. Under the optimized experimental conditions, we achieved a 3.5-fold reduction in nonspecific adsorption of non-target proteins with a 1000-fold enhancement in detection sensitivity of the device for the analysis of human epidermal growth factor receptor 2 (HER2) protein spiked in serum. This approach can be applicable in diverse fields including biosensors, cellular and molecular separation systems and biomedical applications to remove/reduce nonspecific adsorption of molecular and cellular species., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We report a simple electrochemical method referred to as "eMethylsorb" for the detection of DNA methylation. The method relies on the base dependent affinity interaction of DNA with gold. The methylation status of DNA is quantified by monitoring the electrochemical current as a function of the relative adsorption level of bisulphite treated DNA samples onto a bare gold electrode. This method can successfully distinguish methylated and unmethylated epigenotypes at single CpG resolution.

The analysis of DNA methylation is becoming increasingly important both in the clinic and also as a research tool to unravel key epigenetic molecular mechanisms in biology. Current methodologies for the quantification of regional DNA methylation (i.e., the average methylation over a region of DNA in the genome) are largely affected by comprehensive DNA sequencing methodologies which tend to be expensive, tedious, and time-consuming for many applications. Herein, we report an alternative DNA methylation detection method referred to as "Methylsorb", which is based on the inherent affinity of DNA bases to the gold surface (i.e., the trend of the affinity interactions is adenine > cytosine ≥ guanine > thymine).1 Since the degree of gold-DNA affinity interaction is highly sequence dependent, it provides a new capability to detect DNA methylation by simply monitoring the relative adsorption of bisulfite treated DNA sequences onto a gold chip. Because the selective physical adsorption of DNA fragments to gold enable a direct read-out of regional DNA methylation, the current requirement for DNA sequencing is obviated. To demonstrate the utility of this method, we present data on the regional methylation status of two CpG clusters located in the EN1 and MIR200B genes in MCF7 and MDA-MB-231 cells. The methylation status of these regions was obtained from the change in relative mass on gold surface with respect to relative adsorption of an unmethylated DNA source and this was detected using surface plasmon resonance (SPR) in a label-free and real-time manner. We anticipate that the simplicity of this method, combined with the high level of accuracy for identifying the methylation status of cytosines in DNA, could find broad application in biology and diagnostics.

Quantitative and accurate detection of multiple biomarkers would allow for the rapid diagnosis and treatment of diseases induced by pathogens. Monoclonal antibodies are standard affinity reagents applied for biomarkers detection; however, their production is expensive and labor-intensive. Herein, we report on newly developed nanoyeast single-chain variable fragments (NYscFv) as an attractive alternative to monoclonal antibodies, which offers the unique advantage of a cost-effective production, stability in solution, and target-specificity. By combination of surface-enhanced Raman scattering (SERS) microspectroscopy using glass-coated, highly purified SERS nanoparticle clusters as labels, with a microfluidic device comprising multiple channels, a robust platform for the sensitive duplex detection of pathogen antigens has been developed. Highly sensitive detection for individual Entamoeba histolytica antigen EHI&#95;115350 (limit of detection = 1 pg/mL, corresponding to 58.8 fM) and EHI&#95;182030 (10 pg/mL, corresponding 453 fM) with high specificity has been achieved, employing the newly developed corresponding NYscFv as probe in combination with SERS microspectroscopy at a single laser excitation wavelength. Our first report on SERS-based immunoassays using the novel NYscFv affinity reagent demonstrates the flexibility of NYscFv fragments as viable alternatives to monoclonal antibodies in a range of bioassay platforms and paves the way for further applications.

Simple, rapid, and inexpensive strategies for detecting DNA methylation could facilitate routine patient diagnostics. Herein, we describe a microdevice based electrochemical assay for the detection of locus-specific DNA methylation at single CpG dinucleotide resolution after bisulfite conversion of a target DNA sequence. This is achieved by using the ligase chain reaction (LCR) to recognize and amplify a C to T base change at a CpG site and measuring the change electrochemically (eLCR). Unlike other electrochemical detection methods for DNA methylation, methylation specific (MS)-eLCR can potentially interrogate any CpG of interest in the genome. MS-eLCR also distinguishes itself from other electrochemical LCR detection schemes by integrating a peroxidase-mimicking DNAzyme sequence into the LCR amplification probes design which in turn, serves as a redox moiety when bound with a hemin cofactor. Following hybridization to surface-bound capture probes, the DNAzyme-linked LCR products induce electrocatalytic responses that are proportional to the methylation levels of the gene locus in the presence of hydrogen peroxide. The performance of the assay was evaluated by simultaneously detecting C to T changes at a locus associated with cancer metastasis in breast cancer cell lines and serum-derived samples. MS-eLCR required as little as 0.04 pM of starting material and was sensitive to 10-15% methylation change with good reproducibility (RSD=7.9%, n=3). Most importantly, the accuracy of the method in quantifying locus-specific methylation was comparable to both fluorescence-based and Next Generation Sequencing approaches. We thus believe that the proposed assay could potentially be a low cost alternative to current technologies for DNA methylation detection of specific CpG sites., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The time and costs associated with monoclonal antibody production limit the potential for portable diagnostic devices to penetrate the market. Replacing the antibody with a low-cost alternate affinity reagent would reduce the costs of diagnostic development and use, and lead to new portable diagnostic devices towards many diseases. Herein, we present low-cost affinity reagents, nano-yeast-scFv, on commercially available, inexpensive, and portable screen-printed electrodes for the label-free electrochemical detection of Entamoeba histolytica cyst antigens. The biosensor was able to detect antigen at concentrations down to 10 pg mL(-1) in buffer with an inter-assay reproducibility of (% RSD, n=3) 4.1%. The applicability of two differently engineered nano-yeast-scFv to each specifically detect their cognant E. histolytica cyst antigens was demonstrated in a biological matrix derived from human stool. Because of the simple, inexpensive, and sensitive nature of this methodology, it may offer a low-cost alternative to immunosensors based on antibody-target recognition., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This communication reports the use of an electrohydrodynamic surface shear force to selectively manipulate colloid-surface interactions. We demonstrate the selection of strongly (specifically) bound biomolecule-functionalized colloidal beads over more weakly (non-specifically) bound beads using a tuneable alternating current electrohydrodynamic (ac-EHD) force, which drives lateral fluid motion within a few nanometers of an electrode surface. By externally "tuning" the strength of the ac-EHD force, we demonstrate a significant enhancement of capture efficiency for specifically bound colloids, along with a removal of the adsorption of non-specific colloidal beads - a process which may be observed in real-time.

DNA methylation has the potential to be a clinically important biomarker in cancer. This communication reports a real-time and label-free biosensing strategy for DNA methylation detection in the cancer cell line. This has been achieved by using surface plasmon resonance biosensing combined with the highly specific molecular inversion probe based amplification method, which requires only 50 ng of bisulfite treated genomic DNA.

We report a new tuneable alternating current (ac) electrohydrodynamics (ac-EHD) force referred to as “nanoshearing” which involves fluid flow generated within a few nanometers of an electrode surface. This force can be externally tuned via manipulating the applied ac-EHD field strength. The ability to manipulate ac-EHD induced forces and concomitant fluid micromixing can enhance fluid transport within the capture domain of the channel (e.g., transport of analytes and hence increase target–sensor interactions). This also provides a new capability to preferentially select strongly bound analytes over nonspecifically bound cells and molecules. To demonstrate the utility and versatility of nanoshearing phenomenon to specifically capture cancer cells, we present proof-of-concept data in lysed blood using two microfluidic devices containing a long array of asymmetric planar electrode pairs. Under the optimal experimental conditions, we achieved high capture efficiency (e.g., approximately 90%; %RSD=2, n=3) with a 10-fold reduction in nonspecific adsorption of non-target cells for the detection of whole cells expressing Human Epidermal Growth Factor Receptor 2 (HER2). We believe that our ac-EHD devices and the use of tuneable nanoshearing phenomenon may find relevance in a wide variety of biological and medical applications.

We report a tunable alternating current electro-hydrodynamic (ac-EHD) force which drives lateral fluid motion within a few nanometers of an electrode surface. Because the magnitude of this fluid shear force can be tuned externally (e.g., via the application of an ac electric field), it provides a new capability to physically displace weakly (nonspecifically) bound cellular analytes. To demonstrate the utility of the tunable nanoshearing phenomenon, we present data on purpose-built microfluidic devices that employ ac-EHD force to remove nonspecific adsorption of molecular and cellular species. Here, we show that an ac-EHD device containing asymmetric planar and microtip electrode pairs resulted in a 4-fold reduction in nonspecific adsorption of blood cells and also captured breast cancer cells in blood, with high efficiency (approximately 87%) and specificity. We therefore feel that this new capability of externally tuning and manipulating fluid flow could have wide applications as an innovative approach to enhance the specific capture of rare cells such as cancer cells in blood.

Early diagnosis of disease requires highly specific measurement of molecular biomarkers from femto to pico-molar concentrations in complex biological (e.g., serum, blood, etc.) samples to provide clinically useful information. While reaching this detection limit is challenging in itself, these samples contain numerous other non-target molecules, most of which have a tendency to adhere to solid surfaces via nonspecific interactions. Herein, we present an entirely new methodology to physically displace nonspecifically bound molecules from solid surfaces by utilizing a newly discovered "tuneable force", induced by an applied alternating electric field, which occurs within few nanometers of an electrode surface. This methodology thus offers a unique ability to shear-off loosely bound molecules from the solid/liquid interface. Via this approach, we achieved a 5-fold reduction in nonspecific adsorption of non-target protein molecules and a 1000-fold enhancement for the specific capture of HER2 protein in human serum.

Microfabricated devices for the electrochemical detection of single DNA base changes in cancer cell lines are highly desirable due to the inherent advantages such as portability, simplicity, and the rapid and inexpensive nature of electrochemical readout methods. Moreover, molecular sensors that use microscale-footprint working electrodes have shown high signal-to-noise ratio. Herein we report a microdevice-based electrochemical assay (μ-eLCR) measuring ligase chain reaction (LCR)-amplified long and short "knife" motifs which reflect the presence or absence of a DNA base change of interest in a target sequence. This μ-eLCR approach has higher sensitivity (4.4 to 10 fold improvement over macrodisk electrodes) and good reproducibility (%RSD 6.5%, n = 12) for the detection of LCR-amplified DNA bases. The devices also exhibited excellent sensitivity for the detection of DNA methylation (C to T base change in a locus associated with cancer metastasis) in two cell lines and serum derived DNA samples. We believe that the μ-eLCR device may be a useful diagnostic tool for inexpensive and rapid detection of single DNA base change applications such as DNA methylation and single nucleotide polymorphism (SNP) detection.

Inexpensive, simple and quick detection of pathogen antigens in human samples is a key global health objective. Limiting factors include the cost and complexity of diagnostic tests that utilize antibody probes. Herein, we present a method for label-free electrochemical detection of a protein from the enteric pathogen Entamoeba histolytica using cell-free yeast-embedded antibody-like fragments (yeast-scFv) as novel affinity reagents.

Simple, inexpensive and wide-scaled analysis of single DNA base changes ('point mutations' which include SNP's, somatic mutations and epigenetic changes) is one of the holy grail's of point-of-care diagnostics. Herein, we present eLCR: a simple methodology that fuses Ligase Chain Reaction (LCR) with electrochemical detection based on DNA-mediated charge transport. LCR generates long and short "knife" motifs representing the presence or absence of single DNA base changes, which are then detected electrochemically by either methylene blue intercalation or horseradish peroxidase labelling.

An electrochemical immunoassay which minimizes nonspecific protein adsorption and improves detection sensitivity of proteomic cancer biomarker is described. Our technique comprises two novel features: (i) a high density terminally functionalized poly(N-isopropyl acrylamide) 'brush' layer is grown by surface initiated reversible addition fragmentation chain transfer (RAFT) polymerization method from the electrode surface in order to minimize nonspecific adsorption of serum proteins and other biomolecules, and (ii) a signal amplifying 'bionanoconjugate' comprised of graphene oxide nanosheets decorated with CdSe quantum dots and recombinant single-chain variable fragments towards MSLN, is used to 'physically' amplify the anodic stripping voltammetric signal. This method enabled a detection limit of ca. 1 pg/mL MSLN (RSD=4.6%, n=4) spiked in serum samples. Because of the simple, specific and sensitive nature of this methodology, we feel that it may find potential use in serum-based protein diagnostics., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The flow rate dependencies of the aperiodic direct current (dc) and fundamental to eighth alternating current (ac) harmonic components derived from large-amplitude Fourier transformed ac (FT-ac) voltammetry have been evaluated in a microfluidic flow cell containing a 25 μm gold microband electrode. For the oxidation of ferrocenemethanol ([FcMeOH]/[FcMeOH](+) process) in aqueous 0.1 M KNO(3) electrolyte, standard "Levich-like" dc behavior is observed for the aperiodic dc component, which enables the diffusion coefficient for FcMeOH to be obtained. In experimental studies, the first and second ac harmonic components contain contributions from the double layer capacitance current, thereby allowing details of the non-Faradaic current to be established. In contrast, the higher order harmonics and dc aperiodic component are essentially devoid of double layer capacitance contributions allowing the faradaic current dependence on flow rate to be studied. Significantly, flow rate independent data conforming to linear diffusion controlled theory are found in the sixth and higher ac harmonics at a frequency of 15 Hz and for all ac harmonics at a frequency of ≥ 90 Hz. Analysis of FT-ac voltammograms by theory based on stationary microband or planar electrode configurations confirms that stationary microband and planar electrode configurations and experimental data all converge for the higher order harmonics and establishes that the electrode kinetics are very fast (≥1 cms(-1)). The ability to locate, from a single experiment, a dc Faradaic component displaying Levich behavior, fundamental and second harmonics that contain details of the double layer capacitance, and Faradaic ac higher order harmonic currents that are devoid of capacitance, independent of the volume flow rate and also conform closely to mass transport by planar diffusion, provides enhanced flexibility in mass transport and electrode kinetic analysis and in understanding the performance of hydrodynamic electrochemical cells and reactors.

An electrochemical immunosensor for the detection of a cancer biomarker protein in serum at femtomolar concentrations with ultralow background response has been developed, which consists of (i) a hydrophilic polyacrylic acid brush-modified indium tin oxide substrate as an antifouling substrate and (ii) a graphene-quantum dots-antibody 'bionanoconjugate' as a signal amplification label in voltammetric detection of targets in a glassy carbon electrode.

The higher harmonic components available from large-amplitude Fourier-transformed alternating current (FT-ac) voltammetry enable the surface active state of a copper electrode in basic media to be probed in much more detail than possible with previously used dc methods. In particular, the absence of capacitance background current allows low-level Faradaic current contributions of fast electron-transfer processes to be detected; these are usually completely undetectable under conditions of dc cyclic voltammetry. Under high harmonic FT-ac voltammetric conditions, copper electrodes exhibit well-defined and reversible premonolayer oxidation responses at potentials within the double layer region in basic 1.0 M NaOH media. This process is attributed to oxidation of copper adatoms (Cu*) of low bulk metal lattice coordination numbers to surface-bonded, reactive hydrated oxide species. Of further interest is the observation that cathodic polarization in 1.0 M NaOH significantly enhances the current detected in each of the fundamental to sixth FT-ac harmonic components in the Cu*/Cu hydrous oxide electron-transfer process which enables the underlying electron transfer processes in the higher harmonics to be studied under conditions where the dc capacitance response is suppressed; the results support the incipient hydrous oxide adatom mediator (IHOAM) model of electrocatalysis. The underlying quasi-reversible interfacial Cu*/Cu hydrous oxide process present under these conditions is shown to mediate the reduction of nitrate at a copper electrode, while the mediator for the hydrazine oxidation reaction appears to involve a different mediator or active state redox couple. Use of FT-ac voltammetry offers prospects for new insights into the nature of active sites and electrocatalysis at the electrode/solution interface of Group 11 metals in aqueous media.

Since 1992, when the room temperature ionic liquids (ILs) based on the 1-alkyl-3-methylimidazolium cation were reported to provide an attractive combination of an electrochemical solvent and electrolyte, ILs have been widely used in electrodeposition, electrosynthesis, electrocatalysis, electrochemical capacitor, and lithium batteries. However, it has only been in the last few years that electrochemical biosensors based on carbon ionic liquid electrodes (CILEs) and IL-modified macrodisk electrodes have been reported. However, there are still a lot of challenges in achieving IL-based sensitive, selective, and reproducible biosensors for high speed analysis of biological and environmental compounds of interest. This review discusses the principles of operation of electrochemical biosensors based on CILEs and IL/composite-modified macrodisk electrodes. Subsequently, recent developments and major strategies for enhancing sensing performance are discussed. Key challenges and opportunities of IL-based biosensors to further development and use are considered. Emphasis is given to direct electron-transfer reaction and electrocatalysis of hemeproteins and enzyme-modified composite electrodes., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A highly selective and sensitive electrochemical biosensor has been developed that detects DNA hybridization by employing the electrocatalytic activity of ferrocene (Fc) bearing cyclen complexes (cyclen = 1,4,7,10-tetraazacyclododecane, Fc[Zn(cyclen)H(2)O](2)(ClO(4))(4) (R1), Fc(cyclen)(2) (R2), Fc[Zn(cyclen)H(2)O](ClO(4))(2) (R3), and Fc(cyclen) (R4)). A sandwich-type approach, which involves hybridization of a target probe hybridized with the preimmobilized thiolated capture probe attached to a gold electrode, is employed to fabricate a DNA duplex layer. Electrochemical signals are generated by voltammetric interrogation of a Fc bearing Zn-cyclen complexes that selectively and quantitatively binds to the duplex layers through strong chelation between the cyclen complexes and particular nucleobases within the DNA sequence. Chelate formation between R1 or R3 and thymine bases leads to the perturbation of base-pair (A-T) stacking in the duplex structure, which greatly diminishes the yield of DNA-mediated charge transport and displays a marked selectivity to the presence of the target DNA sequence. Coupling the redox chemistry of the surface-bound Fc bearing Zn-cyclen complex and dimethylamine provides an electrocatalytic pathway that increases sensitivity of the assay and allows the 100 fM target DNA sequence to be detected. Excellent selectivity against even single-base sequence mismatches is achieved, and the DNA sensor is stable and reusable.

The oxidative electrochemistry of [CpFe(CO)(2)](2), 1 (Cp = [eta(5)-C(5)H(5)](-)), was examined in detail in ionic liquids (ILs) composed of ions of widely varying Lewis acid-base properties. Cyclic voltammetric responses were strongly dependent on the nucleophilic properties of the IL anion, but all observations are consistent with the initial formation of 1(+) followed by attack from the IL anion. In [NTf(2)](-)-based ILs ([NTf(2)](-) = bis(trifluoromethylsulfonyl)amide), the process shows nearly ideal chemical reversibility as the reaction between 1(+) and [NTf(2)](-) is very slow. This is highly significant, as 1(+) is known to be highly susceptible to nucleophilic attack and its stability indicates a remarkable lack of coordinating ability of these ILs. In 1-methyl-3-butylimidazolium hexafluorophosphate, [bmim][PF(6)], the oxidation of 1 is still largely reversible, but there is more pronounced evidence of [PF(6)](-) coordination. In contrast, 1 exhibits an irreversible two-electron oxidation process in a dicyanamide-based IL. This overall oxidation process is thought to proceed via an ECE mechanism, details of which are presented. Rate constants were estimated by fitting the experimental data to digital simulations of the proposed mechanism. The use of [NTf(2)](-)-based ILs as a supporting electrolyte in CH(2)Cl(2) was examined by using this solvent/electrolyte as a medium in which to perform bulk electrolyses of 1 and 1*, the permethylated analogue [Cp*Fe(CO)(2)](2) (Cp* = [eta(5)-C(5)(CH(3))(5)](-)). These cleanly yielded the corresponding binuclear radical-cation species, 1(+) and 1*(+), which were subsequently characterized by electron paramagnetic resonance (EPR) spectroscopy. In addition to the above oxidation studies, the reduction of 1 was studied in each of the ILs; differences in cathodic peak potentials are attributed, in part, to ion-pairing effects. This study illustrates the wide range of electrochemical environments available with ILs and demonstrates their utility for the investigation of the redox properties of metal carbonyls and other organometallic compounds.

Electrochemical studies on the Fc + e(-) <==> Fc(+) (Fc = ferrocene) process have been undertaken via the oxidation of Fc and reduction of Fc(+) as the hexafluorophosphate (PF(6)(-)) or tetrafluoroborate (BF(4)(-)) salts and their mixtures in three ionic liquids (ILs) (1-butyl-1-methylpyrrolidinium bis[(trifluoromethyl)sulfonyl]imide, 1-butyl-3-methylimidazolium tetrafluoroborate, and 1-butyl-3-methylimidazolium hexafluorophosphate). Data obtained at macro- and microdisk electrodes using conventional dc and Fourier-transformed large-amplitude ac (FT-ac) voltammetry reveal that diffusion coefficients for Fc and Fc(+) differ significantly and are a function of the Fc and Fc(+) concentration, in contrast to findings in molecular solvents with 0.1 M added supporting electrolyte media. Thus, the faradaic currents associated with the oxidation of Fc (Fc(0/+)) and reduction of FcPF(6) or FcBF(4) (Fc(+/0)) when both Fc and Fc(+) are simultaneously present in the ILs differ from values obtained when individual Fc and Fc(+) solutions are used. The voltammetry for both the Fc(0/+) and Fc(+/0) processes exhibited near-Nernstian behavior at a glassy carbon macrodisk electrode and a platinum microdisk electrode, when each process was studied individually in the ILs. As expected, the reversible formal potentials (E(o)') and diffusion coefficients (D) at 23 +/- 1 degrees C were independent of the electrode material and concentration. However, when Fc and FcPF(6) or FcBF(4) were both present, alterations to the mass transport process occurred and apparent D values calculated for Fc and Fc(+) were found to be about 25-39% and 32-42% larger, respectively, than those determined from individual solutions. The apparent value of the double layer capacitance determined by FT-ac voltammetry from individual and mixed Fc and Fc(+) conditions at the GC electrode was also a function of concentration. Double layer capacitance values increased significantly with the concentration of Fc and FcPF(6) or FcBF(4) when species were studied individually or simultaneously, but had a larger magnitude under conditions where both species were present. Variation in the structure of the ILs and hence mobilities of the ionic species, when Fc and FcPF(6) or FcBF(4) are simultaneously present, is considered to be the origin of the nonadditivity of the faradaic currents and variation in capacitance.

Unexpected nonadditivity of currents encountered in the electrochemistry of mixtures of ferrocene (Fc) and cobaltocenium cation (Cc(+)) as the PF(6)(-) salt has been investigated by direct current (dc) and Fourier-transformed alternating current (ac) cyclic voltammetry in two aprotic (1-butyl-3-methylimidazolium tetrafluoroborate and 1-butyl-3-methylimidazolium hexafluorophosphate) and three protic (triethylammonium formate, bis(2-hydroxyethyl)ammonium acetate, and triethylammonium acetate) ionic liquids (ILs). The voltammetry of the individual Fc(0/+) and Cc(+/0) couples always exhibits near-Nernstian behavior at glassy carbon and gold electrodes. As expected for an ideal process, the reversible formal potentials and diffusion coefficients at 23 +/- 1 degrees C in each IL determined from measurement on individual Fc and Cc(+) solutions were found to be independent of electrode material, concentration, and technique used for the measurement. However, when Fc and Cc(+) were simultaneously present, the dc and ac peak currents per unit concentration for the Fc(0/+) and Cc(+/0) processes were found to be significantly enhanced in both aprotic and protic ILs. Thus, the apparent diffusion coefficient values calculated for Fc and Cc(+) were respectively found to be about 25 and 35% larger than those determined individually in the aprotic ILs. A similar change in the Fc(0/+) mass transport characteristics was observed upon addition of tetrabutylammonium hexafluorophosphate (Bu(4)NPF(6)), and the double layer capacitance also varied in distinctly different ways when Fc and Cc(+) were present individually or in mixtures. Importantly, the nonadditivity of Faradaic current is not associated with a change in viscosity or from electron exchange as found when some solutes are added to ILs. The observation that the (1)H NMR T(1) relaxation times for the proton resonance in Cc(+) also are modified in mixed systems implies that specific interaction with aggregates of the constituent IL ionic species giving rise to subtle structural changes plays an important role in modifying the mass transport, double layer characteristics, and dynamics when solutes of interest in this study are added to ILs. Analogous voltammetric changes were not observed in studies in organic solvent media containing 0.1 M added supporting electrolyte. Implications of the nonadditivity of Faradaic and capacitance terms in ILs are considered.

Two simple and fast methods for the extraction of the nitrite ion (NO(2)(-)) from food samples have been developed. The methods were characterized by UV-visible spectroscopic and electrochemical measurements, and their performance for NO(2)(-) extraction was compared with a standard method. The extraction methods yielded relative recoveries between 100 and 120% with good reproducibility of 3.9% (RSD, n = 4) in UV-visible experiments. Microchip electrophoresis with electrochemical detection (MCE-ED) coupled with a copper (3-mercaptopropyl)trimethoxysilane [Cu(II)-MPS] complex-modified carbon paste electrode (CPE) has been employed to detect NO(2)(-) in extracted samples. The Cu(II)-MPS complex was synthesized and characterized by voltammetry, XPS, and FT-IR analyses. Experimental parameters affecting the separation and detection performances of the MCE-ED method were assessed and optimized. The potential for the electrocatalytic reduction of NO(2)(-) for MCE-ED was found to be -190 mV (vs Ag/AgCl). When extracted food samples were analyzed by the MCE-ED method, a reproducible response for the NO(2)(-) reduction (RSD of 4.3%) at the modified-CPE reflected the negligible electrode fouling. A wide dynamic range of 1.0-160 ppm was observed for analyzing standard NO(2)(-) with a sensitivity of 0.05106 ± 0.00141, and the detection limit, based on S/N = 3, was found to be 0.35 ± 0.05 ppm. No apparent interference from NO(3)(-), other inorganic ions, and biological compounds was observed under the optimal experimental conditions. A standard addition method for real samples showed wide concentration ranges of 1.10-155 and 1.2-150 ppm for analyzing NO(2)(-) in ham and sausage samples, respectively.

An amperometric lactate biosensor was developed based on a conducting polymer, poly-5,2'-5',2''-terthiophene-3'-carboxylic acid (pTTCA), and multiwall carbon nanotube (MWNT) composite on a gold electrode. Lactate dehydrogenase (LDH) and the oxidized form of nicotinamide adenine dinucleotide (NAD(+)) were subsequently immobilized onto the pTTCA/MWNT composite film. The modified electrode was characterized by quartz crystal microbalance (QCM), scanning electron microscopy (SEM), and electrochemical experiments. The detection signal was amplified by the pTTCA/MWNT assembly onto which a sufficient amount of enzyme was immobilized and stabilized by the covalent bond formation between the amine groups of enzyme and the carboxylic acid groups of the pTTCA/MWNT film. Experimental parameters affecting the sensor responses, such as applied potential, pH, and temperature, were assessed and optimized. Analytical performances and dynamic ranges of the sensor were determined, and the results showed that the sensitivity, stability, and reproducibility of the sensor improved significantly using pTTCA/MWNT composite film. The calibration plot was linear (r(2)=0.9995) over the range of 5 to 90 microM. The sensitivity was approximately 0.0106 microA/microM, with a detection limit of 1 microM, based on a signal/noise ratio of 3. The applicability of the sensor for the analysis of l-lactate concentration in commercial milk and human serum samples was demonstrated successfully.

We have developed electrochemical DNA and protein sensors on screen-printed electrodes based on the catalytic activity of hydrazine. The sensors use carboxylic acid-functionalized conductive polymer, poly-5,2',5',2''-terthiophene-3'-carboxylic acid (polyTTCA) to make firm immobilization of dendrimer (DEN) through the covalent bond formation between the carboxylic acid groups of polymer and amine groups of dendrimer. The gold nanoparticles (AuNPs) were adsorbed on the remaining amine groups of dendrimer. The thiolated DNA probe or primary antibody was subsequently immobilized on the AuNP-covered dendrimer surfaces. Avidin-labeled hydrazine (Av-Hyd) was then immobilized on the sensor surfaces through the avidin-biotin interaction between the Av-Hyd unit and the biotinylated DNA or secondary antibody. The electrocatalytic reduction current of H(2)O(2) was measured by differential pulse voltammetry. The detection signal was amplified by the polyTTCA/DEN assembly loaded with AuNPs (approximately 3.5 nm) onto which target analyte-linked Av-Hyd was adsorbed. Linear dynamic ranges for the electrocatalytic detection of DNA and human immunoglobulin G (IgG) extending from 50 fM to 7.5 nM and from 40 fg/ml to 2.5 ng/ml, respectively, were observed along with detection limits of approximately 30 fM and 25 fg/ml, respectively. The low detection limit of the disposable sensors offers good promise for practical DNA and protein detection.

A simple and sensitive on-chip preconcentration, separation, and electrochemical detection (ED) method for the electrophoretic analysis of food dyes was developed. The microchip comprised of three parallel channels: the first two are for the field-amplified sample stacking (FASS) and subsequent field-amplified sample injection (FASI) steps, while the third one is for the micellar EKC with ED (MEKC-ED) step. The food dyes were initially extracted from real samples by employing a method that was simpler, easier, and faster compared with a standard method. The extraction of the samples was characterized by UV-Vis and electrochemical experiments. The chronoamperometric detection was performed with a glassy carbon electrode coupled horizontally with the microchip at the separation channel exit. Experimental parameters affecting the analytical performance of the method were assessed and optimized. The sensitivity of the method was improved by approximately 10,800-fold when compared with a conventional MEKC-ED analysis. Reproducible response was observed during multiple injections of samples with an RSD of <7.2% (n=5). The calibration plots were linear (r2=0.998) within the range of 1.0 nM-1.0 microM for all food dyes. LODs were estimated between 1.0 and 5.0 nM, based on S/N=3, for food dyes. The applicability of the method for the analysis of food dyes in real sample was demonstrated.

A sensitive electrochemical assay of DNA and proteins employing electrocatalytic reduction of hydrogen peroxide by labeled hydrazine on the probe immobilized surfaces was developed. The method utilizes a conducting polymer, poly-5, 2':5', 2''-terthiophene-3'-carboxylic acid (pTTCA), covalently linked to the dendrimer (DEN) and hydrazine. The detection signal was amplified by the pTTCA/DEN assembly loaded with Au nanoparticles (particle size, approximately 3.5 nm) onto which huge target DNA- or proteins-linked hydrazine labels (avidin-hydrazine) were adsorbed. The linear dynamic ranges for the electrocatalytic detection of DNA and proteins, extending from 1.0 fM to 10 microM and 10 fg/mL to 10 ng/mL, were observed, along with the detection limits of 450 aM (2700 DNA molecules in a 10-microL sample) and 4.0 fg/mL, respectively. The method eliminates the use of enzymes for DNA and protein detection and opens a way for DNA-free detection of proteins. The simplicity, good reproducibility (RSD, <4.3% for n = 10), and low detection limit of the method offer a good promise for practical DNA and protein analyses.

Label-free detection of bisphenol A based on the impedance measurement was achieved with an impedimetric immunosensor. The immunosensor was fabricated by the covalent bond formation between a polyclonal antibody and a carboxylic acid group functionalized onto a nano-particle comprised conducting polymer. By using a commercial reagent 4,4-bis(4-hydroxyphenyl) valeric acid (BHPVA), which has an analogous structure of BPA, we have prepared the antigen through the conjugation of BHPVA with bovine serum albumin (BSA) and then produced a specific polyclonal antibody. The immobilization of antibody and the interaction between antibody and antigen were studied using quartz crystal microbalance (QCM) and electrochemical impedance spectroscopic (EIS) techniques. The impedance and mass changes due to the specific immuno-interaction at the sensor surface were utilized to detect antigen and bisphenol A (BPA). The immunosensor showed specific recognition of BPA with less interference than 4.5% from other common phenolic compounds. Under an optimized condition, the linear dynamic range of BPA detection was between 1 and 100 ng/ml. The detection limit of bisphenol A was determined to be 0.3+/-0.07 ng/ml. The proposed immunosensor was applied to a human serum sample and the BPA concentration was determined by the standard addition method.

We have developed a simple and sensitive on-chip preconcentration, separation, and electrochemical detection (ED) method for trace analysis of DNA. The microchip comprised of three parallel channels: the first two are for the field-amplified sample stacking and subsequent field-amplified sampled injection steps, while the third one is for the microchip gel electrophoresis (MGE) with ED (MGE-ED). To improve preconcentration and separation performances of the method, the stacking and separation buffers containing the hydroxypropyl cellulose (HPC) matrix were modified with gold nanoparticles (AuNPs). The formation of AuNPs and HPC/AuNP-modified buffers were characterized by UV-visible spectroscopy and TEM experiments. The conducting polymer-modified electrode was also modified with AuNPs to enhance detection performances of the electrode. The conducting polymer/AuNP layers act as electrocatalysts for the direct detection of DNA based on their oxidation in a solution phase. The total sensitivity was improved by approximately 25 000-fold when compared with a conventional MGE-ED analysis. The calibration plots were linear (r2 = 0.9993) within the range of 0.003-1.0 pg/microL for a 20-bp DNA sample. The sensitivity was 0.20 nA/(fg/microL), with a detection limit of 5.7 amol in a 50-microL sample, based on S/N = 3. The applicability of the method for the analysis of 13 fragments present in a 100-bp DNA ladder was successfully demonstrated.

A CE microsystem coupled with a microchip and a copper-(3-mercaptopropyl) trimethoxysilane (Cu-MPS) complex-modified carbon paste electrode (CPE) was developed for the simultaneous analysis of nitrite and nitrate. The method is based on the electrocatalytic reduction of both analytes with the modified electrode. The Cu-MPS complex was characterized by voltammetric, XPS, and FT-IR analyses. Experimental parameters affecting the sensitivity of the modified electrode were assessed and optimized. The best separation was achieved in a 60 mm separation channel filled with a 20 mM acetate buffer of pH 5.0 containing 3.0 mM CTAB at separation field strength of -250 V/cm within 90 s. The detection potential for the simultaneous analysis of nitrite and nitrate was found to be -225 mV versus Ag/AgCl. A reproducible response (RSD of 3.2% (nitrite) and 2.8% (nitrate), n = 8) for repetitive sample injections reflected the negligible electrode fouling at the modified CPE. The interference effect was examined for other inorganic ions and biological compounds. A wide hydrodynamic range between 0.25 and 120 microM was observed for analyzing nitrite and nitrate with the sensitivities of 0.069 +/- 0.003 and 0.065 +/- 0.002 nA/microM, and the detection limits, based on S/N = 3, were found to be 0.09 +/- 0.007 and 0.08 +/- 0.009 microM, respectively. The applicability of the method to water and urine samples analyses was demonstrated.

A micrototal analytical method assembling in-channel preconcentration, separation, and electrochemical detection steps has been developed for trace phenolic compounds. A micellar electrokinetic chromatography separation technique was coupled with two preconcentration steps of field-amplified sample stacking (FASS) and field-amplified sample injection (FASI). An amperometric detection method with a cellulose-dsDNA-modified, screen-printed carbon electrode was applied to detect preconcentrated and separated species at the end of the channel. The microchip was composed of three parallel channels: first, two are for the sample preconcentration using FASS and FASI methods, and the third one is for the separation and electrochemical detection. The modification of the electrode surface improved the detection performance by enhancing the signal-to-noise characteristic without surface fouling of the electrode. The method was examined for the analysis of eight phenolic compounds. Experimental parameters affecting the analytical performance of the method were assessed and optimized. The preconcentration factor was increased by about 5200-fold as compared with a simple capillary zone electrophoretic analysis using the same channel. Reproducible response was observed during multiple injections of samples with a RSD of <8.0%. The calibration plots were shown to be linear (with the correlation coefficient between 0.9913 and 0.9982) over the range of 0.4-600 nM. The sensitivity was between 0.17 +/- 0.001 and 0.48 +/- 0.006 nA/nM, with the detection limit of approximately 100 to approximately 150 pM based on S/N = 3. The applicability of the method to the direct analysis of trace phenolic compounds in water samples was successfully demonstrated.