Páginas: 34 (8448 palabras) Publicado: 25 de agosto de 2010
Laboratory Investigation (2008) 88, 196–206 & 2008 USCAP, Inc All rights reserved 0023-6837/08 $30.00

A novel and simple cell-based detection system with a collagen-encapsulated B-lymphocyte cell line as a biosensor for rapid detection of pathogens and toxins
Pratik Banerjee1, Dominik Lenz2, Joseph Paul Robinson2, Jenna L Rickus3 and Arun K Bhunia1
Cell-based biosensors (CBBs) are becomingimportant tools for biosecurity applications and rapid diagnostics in food microbiology for their unique capability of detecting physiologically hazardous materials. A multi-well plate-based biosensor containing B-cell hybridoma, Ped-2E9, encapsulated in type I collagen matrix, was developed for rapid detection of viable cells of pathogenic Listeria, the toxin listeriolysin O, and the enterotoxinfrom Bacillus species. This sensor measures the alkaline phosphatase release from infected Ped-2E9 cells colorimetrically. Pathogenic L. monocytogenes cells and toxin preparations from L. monocytogenes or B. cereus showed cytotoxicity ranging from 24 to 98% at 3–6 h postinfection. In contrast, nonpathogenic L. innocua (F4247) and B. subtilis induced minimal cytotoxicity, ranging only 0.4–7.6%.Laser scanning cytometry and cryo-nano scanning electron microscopy confirmed the live or dead status of the infected Ped2E9 cells in gel matrix. This paper presents the first example of a cell-based sensing system using collagen-encapsulated mammalian cells for rapid detection of pathogenic bacteria or toxin, and demonstrates a potential for onsite use as a portable detection system.
LaboratoryInvestigation (2008) 88, 196–206; doi:10.1038/labinvest.3700703; published online 3 December 2007

KEYWORDS: cell-based sensor; collagen entrapment; Listeria monocytogenes; Bacillus cereus; Ped-2E9 cells; rapid diagnostics

Detection of biological entities using cell-based assays (CBAs) has already found its niche in biomedical research as well as in clinical, environmental, and bioprocessindustries.1–5 The rapid detection methodologies applicable to these fields using CBAs have had enormous impact on the emergence of cellbased biosensors (CBBs).5–8 In a CBB, the normal physiological response of a living cell or cellular component resulted by one or more external factors (stimuli) is recorded.9,10 This ‘response’ can be qualitatively or quantitatively analyzed to deduce the nature andcharacter of the external ‘stimuli’.5,6,8–11 In some of these CBBs the sensing elements could be the vegetative cells of bacteria,5,12 or eukaryotic13 or mammalian cells.8,14,15 Different types of platforms or materials have been used to ‘pattern’ or lay different types of higher eukaryotic cells to design cell-based sensors.5,10,16–20 Interfacial engineering to couple biological cell (or cellularcomponents) to signal transducers or detection platforms is critical for successful cell-based biosensing.21–24 The types of materials used for immobilization and entrapment of living cells vary

widely, encompassing biocompatible polymers (such as polyethylene glycol, alginate, synthetic peptides or silica gel)25–28 to biologically derived substances such as collagen or fibronectin, to name afew.29,30 The viability of the living cell after immobilization determines the actual working life of the sensor. For that reason, materials that best mimic the actual physiological conditions would be the most effective material to use as a matrix for cell harboring. Cellular entrapment in biomaterials such as collagen has distinct advantages, including the unparalleled biocompatibility ofcollagen, which makes it one of the most versatile biomaterials used for tissue culture known so far.31,32 In addition, collagen gel can be prepared with different pore sizes that trap different size cells by varying the concentration.33,34 Collagen gel has high mechanical strength and has excellent gelling properties.33 Three-dimensional (3D) encapsulation of different cell types in collagen gel has...
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