Muhammad J. A. Shiddiky

Application of ionic liquids in electrochemical sensing systems

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.

Detecting Exosomes Specifically: A Multiplexed Device Based on Alternating Current Electrohydrodynamic Induced Nanoshearing

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.

Nonadditivity of Faradaic currents and modification of capacitance currents in the voltammetry of mixtures of ferrocene and the cobaltocenium cation in protic and aprotic ionic liquids.

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.

Graphene/quantum dot bionanoconjugates as signal amplifiers in stripping voltammetric detection of EpCAM biomarkers

A sensitive electrochemical immunosensor for the detection of epithelial cell adhesion molecule (EpCAM) antigen, a common marker for tumors of epithelial origin, employing bionanoconjugates as signal-transduction labels has been developed. The bionanoconjugates were fabricated by carboxylation of the two-dimensional graphene oxide nanosheets (GRs) and immobilizing streptavidin and amine-functionalized CdSe quantum dots (QDs) on carboxylated GRs via carbodiimide coupling chemistry, followed by the immunoreaction with the biotinylated secondary antibodies. Since carboxylated GRs have a higher density of active sites, it allows a large number of CdSe QDs to be immobilized onto the surface of the bionanoconjugates, and hence, enhance the sensitivity of the immunosensor. The method enabled detection limits of 100 fg/mL and 1 pg/mL (based on the S/N=3) in PBS buffer and serum samples, respectively, using anodic stripping voltammetric readout. The immunosensor showed a good selectivity, reproducibility, and long-storage stability, and may become a promising technique for the early detection of tumor biomarker in clinical/biological samples.

Enabling Rapid and Specific Surface-Enhanced Raman Scattering Immunoassay Using Nanoscaled Surface Shear Forces

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.

Highly Selective and Sensitive DNA Assay Based on Electrocatalytic Oxidation of Ferrocene Bearing Zinc(II)-Cyclen Complexes with Diethylamine

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.

Duplex Microfluidic SERS Detection of Pathogen Antigens with Nanoyeast Single-Chain Variable Fragments

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_115350 (limit of detection = 1 pg/mL, corresponding to 58.8 fM) and EHI_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.

DNA–bare gold affinity interactions: mechanism and applications in biosensing

The adsorption of DNA onto gold due to affinity interactions is highly desirable for developing low-cost, convenient and sensitive biosensors. To date, DNA–gold adsorption phenomenon has been demonstrated as one of the most promising physical mechanisms for achieving precise control over unmodified gold nanoparticles (AuNPs) aggregation, and DNA monolayer formation on gold surfaces. The adsorption phenomenon is exquisitely controlled by many factors including intermolecular forces, along with DNA composition and sequence. The understanding and manipulation of these factors have allowed broad biosensing applications and notably, sequence-dependent DNA–gold adsorption which may be highly relevant for DNA methylation detection in cancer. Herein, we review the underlying principles governing DNA–gold adsorption as well as recent biosensing strategies based on differential ssDNA/dsDNA–AuNPs adsorption and sequence-dependent DNA–gold adsorption. Finally, we have also contributed insights regarding the future trend of DNA–gold adsorption-based biosensors.

Label-free electrochemical detection of an Entamoeba histolytica antigen using cell-free yeast-scFv probes

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.

Electrophoretic analysis of food dyes using a miniaturized microfluidic system

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 ∼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 μM 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.