Nano-yeast–scFv probes on screen-printed gold electrodes for detection of Entamoeba histolytica antigens in a biological matrix

Grewal, Yadveer S.; Shiddiky, Muhammad J. A.; Spadafora, Lauren J.; Cangelosi, Gerard A.; Trau, Matt

doi:10.1016/j.bios.2013.12.043

Cited by 39 publications

(63 citation statements)

References 28 publications

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“…Screen-printed electrodes have been shown to be ideal for developing point-of-care assays due to their low cost, disposability and design exibility as compared to traditional electrode materials. [20][21][22] While various assays have been shown to integrate SPE-Au into electrochemical biosensors, 23 its application in DNA methylation detection has yet to be demonstrated.…”

Section: Introductionmentioning

confidence: 99%

eMethylsorb: rapid quantification of DNA methylation in cancer cells on screen-printed gold electrodes

Koo

Sina

Carrascosa

et al. 2014

Analyst

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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 ] 3À 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 ¼ 5.8%, n ¼ 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.

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Section: Introductionmentioning

confidence: 99%

eMethylsorb: rapid quantification of DNA methylation in cancer cells on screen-printed gold electrodes

Koo

Sina

Carrascosa

et al. 2014

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“…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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“…Initial voltammograms obtained from cyclic voltammetry experiments run over the potential range of -0.3 V to +0.3 V at a scan rate of 25 mV/s with respect to the Ag -pseudo-reference electrode initially yielded a low signal-to-noise ratio (SNR), possibly due to endogenous phosphatase activity at the yeast cell surface (see supplementary S3). Sodium azide, a biocide and a preservative [22] was added to halt the cellular metabolism by inhibiting the adenosine triphosphate (ATP) synthesis and the yeast respiration is completely inhibited at 0.15 mM concentrations (~ 0.001%) [35]. It also reacts with carbonyl complexes similar to CO coordinated molecules, thereby inhibiting many yeast metabolites from contributing to unwanted background noise [36].…”

Section: Electrochemical Sandwich Elisamentioning

confidence: 99%

“…Genetically engineered yeast capable of single chain or single domain antibody surface-display have been previously reported [22], and have multiple advantages over classic technologies, since living systems are inherently self-replicating and are often resistant to harsh environmental conditions [10], [22]. Thus there are practical and cost advantages to such constructs.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, integration of BioBrick technology into assay design has the potential to significantly reduce the cost of mass producing detection molecules, as growing a culture of S. cerevisae is far less expensive than current in vivo and in vitro antibody production techniques [25]. Recently, a POC platform to detect E. histolytica antigen using nano-yeast-scFv with labelfree electrochemical detection has been reported in the literature [22], [26]. These data support the use of yeast reagents for low-cost assays, yet leave a number of directions to be further explored and optimized.…”

Section: Introductionmentioning

confidence: 99%

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Yeast dual-affinity biobricks: Progress towards renewable whole-cell biosensors

Venkatesh

Sun

Brickner

et al. 2015

Biosensors and Bioelectronics

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Abstract:Point-of-care (POC) diagnostic biosensors offer a promising solution to improve healthcare, not only in developed parts of the world, but also in resource limited areas that lack adequate medical infrastructure and trained technicians. However, in remote and resource limited settings, cost and storage of traditional POC immunoassays often limit actual deployment. Synthetically engineered biological components ("BioBricks") provide an avenue to reduce costs and simplify assay procedures. In this article, the design and development of an ultra-low cost, whole-cell "renewable" capture reagent for use in POC diagnostic applications is described. Yeast cells were genetically modified to display both single chain variable fragment (scFv) antibodies and goldbinding peptide (GBP) on their surfaces for simple one step enrichment and surface functionalization. Electrochemical impedance spectroscopy (EIS) and fluorescent imaging were used to verify and characterize the binding of cells to gold electrodes. A complete electrochemical detection assay was then performed on screen-printed electrodes fixed with yeast displaying scFv directed to Salmonella outer membrane protein D (OmpD). Electrochemical assays were optimized and cross-validated with established fluorescence techniques. Nanomolar detection limits were observed for both formats.

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Nano-yeast–scFv probes on screen-printed gold electrodes for detection of Entamoeba histolytica antigens in a biological matrix

Cited by 39 publications

References 28 publications

eMethylsorb: rapid quantification of DNA methylation in cancer cells on screen-printed gold electrodes

eMethylsorb: rapid quantification of DNA methylation in cancer cells on screen-printed gold electrodes

Intestinal Amebae

Yeast dual-affinity biobricks: Progress towards renewable whole-cell biosensors

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