Mahmoud Labib

Electrochemical Methods for the Analysis of Clinically Relevant Biomolecules

Rapid progress in identifying biomarkers that are hallmarks of disease has increased demand for high-performance detection technologies. Implementation of electrochemical methods in clinical analysis may provide an effective answer to the growing need for rapid, specific, inexpensive, and fully automated means of biomarker analysis. This Review summarizes advances from the past 5 years in the development of electrochemical sensors for clinically relevant biomolecules, including small molecules, nucleic acids, and proteins. Various sensing strategies are assessed according to their potential for reaching relevant limits of sensitivity, specificity, and degrees of multiplexing. Furthermore, we address the remaining challenges and opportunities to integrate electrochemical sensing platforms into point-of-care solutions.

Three-Mode Electrochemical Sensing of Ultralow MicroRNA Levels

MicroRNAs (miRNAs) are an emerging class of biomarkers that are frequently deregulated in cancer cells and have shown great promise for cancer classification and prognosis. In this work, we developed a three-mode electrochemical sensor for detection and quantitation of ultralow levels of miRNAs in a wide dynamic range of measured concentrations. The sensor facilitates three detection modalities based on hybridization (H-SENS), p19 protein binding (P-SENS), and protein displacement (D-SENS). The combined three-mode sensor (HPD-SENS) identifies as low as 5 aM or 90 molecules of miRNA per 30 μL of sample without PCR amplification, and can be operated within the dynamic range from 10 aM to 1 μM. The HPD sensor is made on a commercially available gold nanoparticles-modified electrode and is suitable for analyzing multiple miRNAs on a single electrode. This three-mode sensor exhibits high selectivity and specificity and was used for sequential analysis of miR-32 and miR-122 on one electrode. In addition, the H-SENS can recognize miRNAs with different A/U and G/C content and distinguish between a fully matched miRNA and a miRNA comprising either a terminal or a middle single base mutation. Furthermore, the H- and P-SENS were successfully employed for direct detection and profiling of three endogenous miRNAs, including hsa-miR-21, hsa-miR-32, and hsa-miR-122 in human serum, and the sensor results were validated by qPCR.

Ferrocene-peptido conjugates: From synthesis to sensory applications

The field of chemical and biological sensing is increasingly dependent on the availability of new functional materials that enhance the ability of the system to respond to chemical interactions. Organometallic bioconjugates derived from amino acids, peptides, proteins, peptide nucleic acids, and dendrimers have had a profound effect in this area and have endowed modern sensory systems with a superior performance. Owing to their fairly high stability, solubility in various solvents, and excellent redox properties, ferrocene and ferrocenyl conjugates have emerged as one of the most important classes of materials that enable direct observation of molecular interactions and as electron mediators. The low potential, reversible redox behavior of the ferrocene/ferrocenium couple is a unique property that finds widespread application in the design of sensory platforms. Currently, there is significant drive to exploit new organometallic systems, in which the presence of ferrocene acting as a redox center is critical and allows the design of highly sensitive electrochemical sensors for the sensing and recognition of a vast array of analytes.

Aptamer-Based Viability Impedimetric Sensor for Bacteria

The development of an aptamer-based viability impedimetric sensor for bacteria (AptaVISens-B) is presented. Highly specific DNA aptamers to live Salmonella typhimurium were selected via the cell-systematic evolution of ligands by exponential enrichment (SELEX) technique. Twelve rounds of selection were performed; each comprises a positive selection step against viable S. typhimurium and a negative selection step against heat killed S. typhimurium and a mixture of related pathogens, including Salmonella enteritidis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Citrobacter freundii to ensure the species specificity of the selected aptamers. The DNA sequence showing the highest binding affinity to the bacteria was further integrated into an impedimetric sensor via self-assembly onto a gold nanoparticle-modified screen-printed carbon electrode (GNP-SPCE). Remarkably, this aptasensor is highly selective and can successfully detect S. typhimurium down to 600 CFU mL(-1) (equivalent to 18 live cells in 30 μL of assay volume) and distinguish it from other Salmonella species, including S. enteritidis and S. choleraesuis. This report is envisaged to open a new venue for the aptamer-based viability sensing of a variety of microorganisms, particularly viable but nonculturable (VBNC) bacteria, using a rapid, economic, and label-free electrochemical platform.

Beyond the Capture of Circulating Tumor Cells: Next-Generation Devices and Materials

Over the last decade, significant progress has been made towards the development of approaches that enable the capture of rare circulating tumor cells (CTCs) from the blood of cancer patients, a critical capability for noninvasive tumor profiling. These advances have leveraged new insights in materials chemistry and microfluidics and allowed the capture and enumeration of CTCs with unprecedented sensitivity. However, it has become increasingly clear that simply capturing and counting tumor cells launched into the bloodstream may not provide the information needed to advance our understanding of the biology of these rare cells, or to allow us to better exploit them in medicine. A variety of advances have now emerged demonstrating that more information can be extracted from CTCs with next-generation devices and materials featuring tailored physical and chemical properties. In this Minireview, the last ten years of work in this area will be discussed, with an emphasis on the groundbreaking work of the last five years, during which the focus has moved beyond the simple capture of CTCs and gravitated towards approaches that enable in-depth analysis.

Aptamer-based viability impedimetric sensor for viruses.

The development of aptamer-based viability impedimetric sensor for viruses (AptaVISens-V) is presented. Highly specific DNA aptamers to intact vaccinia virus were selected using cell-SELEX technique and integrated into impedimetric sensors via self-assembly onto a gold microelectrode. Remarkably, this aptasensor is highly selective and can successfully detect viable vaccinia virus particles (down to 60 virions in a microliter) and distinguish them from nonviable viruses in a label-free electrochemical assay format. It also opens a new venue for the development of a variety of viability sensors for detection of many microorganisms and spores.

Aptamer-based impedimetric sensor for bacterial typing.

The development of an aptamer-based impedimetric sensor for typing of bacteria (AIST-B) is presented. Highly specific DNA aptamers to Salmonella enteritidis were selected via Cell-SELEX technique. Twelve rounds of selection were performed; each comprises a positive selection step against S. enteritidis and a negative selection step against a mixture of related pathogens, including Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Citrobacter freundii, to ensure the species-specificity of the selected aptamers. After sequencing of the pool showing the highest binding affinity to S. enteritidis, a DNA sequence of high affinity to the bacteria was integrated into an impedimetric sensor via self-assembly onto a gold nanoparticles-modified screen-printed carbon electrode (GNPs-SPCE). Remarkably, this aptasensor is highly selective and can successfully detect S. enteritidis down to 600 CFU mL(-1) (equivalent to 18 CFU in 30 μL assay volume) in 10 min and distinguish it from other Salmonella species, including S. typhimurium and S. choleraesuis. This report is envisaged to open a new venue for the aptamer-based typing of a variety of microorganisms using a rapid, economic, and label-free electrochemical platform.

Four-way junction formation promoting ultrasensitive electrochemical detection of microRNA.

MicroRNAs (miRNAs) represent a class of biomarkers that are frequently deregulated in cancer cells and have shown a great promise for cancer classification and prognosis. Here, we endeavored to develop a DNA four-way junction based electrochemical sensor (4J-SENS) for ultrasensitive miRNA analysis. The developed sensor can be operated within the dynamic range from 10 aM to 1 fM and detect as low as 2 aM of miR-122 (∼36 molecules per sample), without PCR amplification. Furthermore, the 4J-SENS was employed to profile endogenouse hsa-miR-122 in healthy human and chronic lymphocyitc leukemia (CLL) patient serum, and the results were validated by qPCR analysis.

Electrochemical sensing of aptamer-facilitated virus immunoshielding.

Oncolytic viruses (OVs) are promising therapeutics that selectively replicate in and kill tumor cells. However, repetitive administration of OVs provokes the generation of neutralizing antibodies (nAbs) that can diminish their anticancer effects. In this work, we selected DNA aptamers against an oncolytic virus, vesicular stomatitis virus (VSV), to protect it from nAbs. A label-free electrochemical aptasensor was used to evaluate the degree of protection (DoP). The aptasensor was fabricated by self-assembling a hybrid of a thiolated ssDNA primer and a VSV-specific aptamer. Electrochemical impedance spectroscopy was employed to quantitate VSV in the range of 800-2200 PFU and a detection limit of 600 PFU. The aptasensor was also utilized for evaluating binding affinities between VSV and aptamer pools/clones. An electrochemical displacement assay was performed in the presence of nAbs and DoP values were calculated for several VSV-aptamer pools/clones. A parallel flow cytometric analysis confirmed the electrochemical results. Finally, four VSV-specific aptamer clones, ZMYK-20, ZMYK-22, ZMYK-23, and ZMYK-28, showed the highest protective properties with dissociation constants of 17, 8, 20, and 13 nM, respectively. Another four sequences, ZMYK-1, -21, -25, and -29, exhibited high affinities to VSV without protecting it from nAbs and can be further utilized in sandwich assays. Thus, ZMYK-22, -23, and -28 have the potential to allow efficient delivery of VSV through the bloodstream without compromising the patients immune system.

Electrochemical analysis of HIV-1 reverse transcriptase serum level: Exploiting protein binding to a functionalized nanostructured surface

This manuscript describes an electrochemical approach to the detection of the reverse transcriptase of the human immunodeficiency virus type-1 (HIV-1 RT) in serum exploiting an organometallic peptide conjugate that is chemically linked to a nanostructured gold surface. The assay format is based on the formation of a thin film of a ferrocene-labeled lipoic acid (Fc-LA) onto a gold nanoparticles-functionalized screen-printed carbon electrode (GNPs-SPCE). Time-of-Flight secondary ion mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy were employed to confirm the binding of the Fc-LA to the electrode surface via formation of a gold-thiol bond. The RT biosensor was developed by covalent attachment of the peptide VEAIIRILQQLLFIH to the carboxylic acid group of Fc-LA. Square wave voltammetry offered a two-dimensional measurement of RT based on the anodic shift and reduction of current density of the Fc redox signal upon binding of RT to its specific peptide. This allowed a linear quantification of the target RT in the range of 1-500 pg mL(-1) equivalent to 0.9-427 fM, with a detection limit of 0.8 pg mL(-1) (0.7 fM) with a short response time.