Department of Animal Science, University of Wyoming , Laramie, Wyoming 82071, United States.
Anal Chem. 2017 Mar 21;89(6):3613-3621. doi: 10.1021/acs.analchem.6b05009. Epub 2017 Mar 7.
The development of transparency-based electrochemical and paper-based colorimetric analytic detection platforms is presented as complementary methods for food and waterborne bacteria detection from a single assay. Escherichia coli and Enterococcus species, both indicators of fecal contamination, were detected using substrates specific to enzymes produced by each species. β-galactosidase (β-gal) and β-glucuronidase (β-glucur) are both produced by E. coli, while β-glucosidase (β-gluco) is produced by Enterococcus spp. Substrates used produced either p-nitrophenol (PNP), o-nitrophenol (ONP), or p-aminophenol (PAP) as products. Electrochemical detection using stencil-printed carbon electrodes (SPCEs) was found to provide optimal performance on inexpensive and disposable transparency film platforms. Using SPCEs, detection limits for electrochemically active substrates, PNP, ONP, and PAP were determined to be 1.1, 2.8, and 0.5 μM, respectively. A colorimetric paper-based well plate system was developed from a simple cardboard box and smart phone for the detection of PNP and ONP. Colorimetric detection limits were determined to be 81 μM and 119 μM for ONP and PNP respectively. While colorimetric detection methods gave higher detection limits than electrochemical detection, both methods provided similar times to positive bacteria detection. Low concentrations (10 CFU/mL) of pathogenic and nonpathogenic E. coli isolates and (10 CFU/mL) E. faecalis and E. faecium strains were detected within 4 and 8 h of pre-enrichment. Alfalfa sprout and lagoon water samples served as model food and water samples, and while water samples did not test positive, sprout samples did test positive within 4 h of pre-enrichment. Positive detection of inoculated (2.3 × 10 and 3.1 × 10 CFU/mL or g of E. coli and E. faecium, respectively) sprout and water samples tested positive within 4 and 12 h of pre-enrichment, respectively.
呈现了基于透明度的电化学和基于纸的比色分析检测平台的发展，这些平台作为从单一分析中检测食源性和水源性细菌的互补方法。使用针对每种细菌产生的酶的底物检测了粪便污染指示物大肠杆菌和肠球菌属。β-半乳糖苷酶（β-gal）和β-葡萄糖醛酸酶（β-glucur）均由大肠杆菌产生，而β-葡萄糖苷酶（β-gluco）则由肠球菌属产生。使用的底物产生要么对硝基苯酚（PNP）、邻硝基苯酚（ONP）或对氨基酚（PAP）作为产物。发现使用模版印刷碳电极（SPCEs）进行电化学检测在廉价且一次性的透明度薄膜平台上提供了最佳性能。使用 SPCEs，确定电化学活性底物 PNP、ONP 和 PAP 的电化学检测限分别为 1.1、2.8 和 0.5 μM。从简单的纸箱和智能手机开发了一种比色纸基孔板系统，用于检测 PNP 和 ONP。比色检测限分别确定为 ONP 和 PNP 的 81 μM 和 119 μM。虽然比色检测方法的检测限高于电化学检测，但两种方法都提供了类似的阳性细菌检测时间。在预富集 4 和 8 小时内，检测到低浓度（10 CFU/mL）的致病性和非致病性大肠杆菌分离株和（10 CFU/mL）肠球菌属和肠球菌属菌株。苜蓿芽和泻湖水样作为模型食物和水样，虽然水样未呈阳性，但在预富集 4 小时内，芽样呈阳性。接种（2.3×10 和 3.1×10 CFU/mL 或 g 的大肠杆菌和肠球菌属，分别）的芽和水样本的阳性检测在预富集 4 和 12 小时内分别呈阳性。
