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

Grewal, Yadveer S.; Shiddiky, Muhammad J. A.; Gray, Sean A.; Weigel, Kris M.; Cangelosi, Gerard A.; Trau, Matt

doi:10.1039/c2cc38882k

Cited by 61 publications

(61 citation statements)

References 15 publications

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“…1 describes an assembly scheme of the biosensor. As reported previously, 18 biotin labeled BSA is used for coating the gold electrode surface. Subsequently, multivalent streptavidin was used as a linker to immobilize the biotin labeled SNA lectin to form a bio-recognition layer on the electrode surface.…”

Section: Resultsmentioning

confidence: 99%

“…[11][12][13][14][15] Among many EC methods, faradaic electrochemical impedance spectroscopy (F-EIS) is one of the most effective methods for the label-free detection of biomolecules and for probing the build-up of the biomaterial sensing lm on the electrodes. [16][17][18] In F-EIS, the binding of a target protein with its ligand (i.e., a specic antibody) on the electrode surface can be detected, where the change in impedance of the electrode surface and its interface to the electrolyte solution containing a redox probe (e.g. [Fe(CN) 6 ] 3À/4À ) is measured in the form of its electron transfer resistance (R ct ).…”

Section: Introductionmentioning

confidence: 99%

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Electrochemical detection of glycan and protein epitopes of glycoproteins in serum

Shah

Hill

Shiddiky

et al. 2014

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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 labelfree voltammetric or impedimetric interrogation of a ferro/ferricyanide redox couple (e.g. [Fe(CN) 6 ] 3À/4À ) 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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Electrochemical detection of glycan and protein epitopes of glycoproteins in serum

Shah

Hill

Shiddiky

et al. 2014

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“…However, the whole-cell yeast scFv molecules are insoluble, often too large for diagnostic application, and they need a labeled secondary antibody to detect the specific antigen. Recently, the same group of investigators further improved the system by using a cell-wall fragments of selected scFv clones, 94 and then made it a label-free antigen detection system. 95 This method is rapid and far more cost-effective than that of mAb production in mice, and shows promise as an effective alternative to traditional mAbs.…”

Section: Single-chain Fragment-variable Probesmentioning

confidence: 99%

Intestinal Amebae

Ali¹

2015

Clinics in Laboratory Medicine

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Among the Entamoeba species that infect humans, Entamoeba histolytica causes diseases, Entamoeba dispar is a harmless commensal, Entamoeba moshkovskii seems to be a pathogen, and the pathogenicity of Entamoeba bangladeshi remains to be investigated. Species-specific detection needed for treatment decisions and for understanding the epidemiology and pathogenicity of these amebae. Antigen-based detection methods are needed for E dispar, E moshkovskii, and E bangladeshi; and molecular diagnostic test capable of detecting E histolytica, E dispar, E moshkovskii, and E bangladeshi simultaneously in clinical samples. Next-generation sequencing of DNA from stool is needed to identify novel species of Entamoeba.

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“…24,25 This strategy is an alternative to the standard approach of solubilizing scFvs prior to anchoring them onto a surface. 99 Importantly, this approach maintains scFvs in the environment in which they were selected to function.…”

Section: Yeast Display Library For Scfv Selectionmentioning

confidence: 99%

Nanoyeast and Other Cell Envelope Compositions for Protein Studies and Biosensor Applications

Grewal

Shiddiky

Mahler

et al. 2016

ACS Appl. Mater. Interfaces

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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.

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

Cited by 61 publications

References 15 publications

Electrochemical detection of glycan and protein epitopes of glycoproteins in serum

Electrochemical detection of glycan and protein epitopes of glycoproteins in serum

Intestinal Amebae

Nanoyeast and Other Cell Envelope Compositions for Protein Studies and Biosensor Applications

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