Novel SERS substrate-based method for quantitatively testing pathogenic bacteria

A technology for quantitative detection and pathogenic bacteria, which is applied in measuring devices, instruments, material analysis by optical means, etc., can solve the problems of low detection sensitivity, cumbersome and time-consuming process, and long growth time, so as to achieve high sensitivity and improved Capture efficiency and specificity

Inactive Publication Date: 2017-05-10
SOUTH CHINA NORMAL UNIVERSITY
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  • Abstract
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Problems solved by technology

[0003] The current gold standard for the detection of Escherichia coli O157:H7 is the traditional isolation and identification method. This method needs to go through steps such as pre-enrichment, selective enrichment, isolation and culture, physiological and biochemical identification, and serotype identification. The whole process is tedious and time-consuming (3~ 7 days), and the whole process needs to be operated by professionals, so this method can only be used by law enforcement agencies to analyze the cause of poisoning incidents and evaluate the food safety situation in the market; immunology, especially immunochromatography methods, do not need to be complicated Instruments and equipment, the detection time is fast and easy to operate, but the overall detection sensitivity of this type of method is low at present (the bacterial concentration needs to reach 10 5 ~10 6 CFU / mL), so for the detection of real samples, a longer enrichment time is often required

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  • Novel SERS substrate-based method for quantitatively testing pathogenic bacteria
  • Novel SERS substrate-based method for quantitatively testing pathogenic bacteria
  • Novel SERS substrate-based method for quantitatively testing pathogenic bacteria

Examples

Experimental program
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Effect test

Embodiment 1

[0053] (1), preparation of gold nanoparticles

[0054] Under constant stirring, 100 mL of 1 mM chloroauric acid (HAuCl 4 ) solution was heated to boiling, and then 6 mL of 38.8 mM trisodium citrate aqueous solution was added. At this time, the color change of the solution is: light yellow-colorless-black-purple-dark red, wait for the solution to turn dark red and continue to heat and reflux for 15-20min. Finally cooled to normal temperature, the preparation obtains the gold nanoparticle of 30nm (such as figure 2 C), figure 2 A is gold nanoparticles obtained under ideal conditions.

[0055] (2), preparation of silica-coated gold nanoparticles

[0056] Add 1mL gold nanoparticles (concentration is about 1mmol / L) to six 1.5mL EP tubes respectively, then add 30μL 0.01mM 4,4′-bipyridine solution in turn, mix well and react for 10min, centrifuge at 8000rpm for 10min, Remove the supernatant and resuspend with 1 mL triple distilled water. Then all the colloids in the 6 EP tubes...

Embodiment 2

[0069] Take 1 mL of the prepared gold nanoparticle solution, put it into an EP tube, add 30 μL of 0.01 mM 4,4′-bipyridine solution, react at room temperature for 10 min, centrifuge at 8000 rpm (10 min), remove the supernatant, and wash with BB buffer Resuspend, add 5μL 0.5mg / mL rabbit anti-E.coli O157:H7, incubate for 2h, centrifuge (8000rpm, 10min) to remove the supernatant, resuspend in BB buffer, then add 30μL 2.5% BSA to block unbound site, centrifuged (8000rpm, 10min) after 1h to remove the supernatant, and resuspended with PBS buffer to prepare bare gold signal probes. Place it with the signal probe in embodiment 1 step (3) for the same time, and measure the Raman signal intensity (such as Figure 5 ). from Figure 5 According to the report, preliminary studies have shown that this new type of SERS substrate has the following advantages: 1) the silicon dioxide silicon layer can ensure that gold nanoparticles are not disturbed by external chemical conditions; The inner...

Embodiment 3

[0071] Take the water body sample of the Guangzhou section of the Pearl River, filter, centrifuge, and dilute 40 times with triple distilled water to eliminate the influence of the matrix (the concentration of E.coli O157:H7 in the water body dilution of the known concentration is 10 2 、10 4 、10 6 、10 8 CFU / mL). According to the Raman spectrum signal intensity obtained in the actual water sample, from Figure 4 Read the corresponding concentration of E.coliO157:H7 from the standard curve in , and multiply it by the corresponding dilution factor to get the actual concentration of E.coli O157:H7 in the sample to be tested. The concentration of the actual water sample obtained according to the linear equation in Example 1 is shown in Table 1 below.

[0072] E.coli O157:H7 concentration and the E.coliO157:H7 concentration measured by the method of this embodiment in the actual water body sample of table 1

[0073] Known Concentration (CFU / mL) Measured concentratio...

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Abstract

The invention discloses a novel SERS substrate-based method for quantitatively testing pathogenic bacteria, and belongs to the technical field of biological detection. Silicon dioxide treated by an immunospectral marker is coated with gold nanoparticles for quantitative test of the pathogenic bacteria. Raman spectra signal monitoring data are more stable and reliable and can be greatly repeated. The method is good in test stability, high in sensitivity, high in specificity, the detection process is simple, fast, accurate and quantitative, and a wide detection interval and a low detection limit are obtained. The method is simple in operation, an SERS immune signal probe and a capture probe can be prepared in advance, the capture probe and the signal probe only need to be sequentially added to a to-be-detected sample for reaction during operation and the test is carried out at once through magnetic separation after the reaction is completed: the collection time is 30s, and then the concentration is immediately read from a standard curve, thereby achieving fast quantitatively testing. The method can meet the requirements of food safety and environment monitoring departments, and has wide practicability.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for quantitatively detecting pathogenic bacteria based on a novel SERS substrate. Background technique [0002] In recent years, foodborne diseases caused by microorganisms have become a worldwide public health hotspot. Among food-borne pathogens, Escherichia coli (Escherichia coli) O157:H7 is the most dangerous one, and is currently recognized as one of the three most important food-borne pathogens in the world. The clinical symptoms of Escherichia coli O157:H7 infection include diarrhea, hemorrhagic enteritis (HC), hemolytic uremic syndrome (HUS) and thrombocytopenic purpura due to thrombosis. Pathogenic Escherichia coli can cause disease outbreaks by contaminating drinking water and food, and seriously endanger human health. [0003] The current gold standard for the detection of Escherichia coli O157:H7 is the traditional isolation and iden...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/65
CPCG01N21/658
Inventor 胡勇军朱挺锋杨康余萌江宁静张文建
Owner SOUTH CHINA NORMAL UNIVERSITY
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