Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Whole-cell biosensor for detecting heavy metal ions in water-soluble samples and its construction and application

A heavy metal ion, biosensor technology, applied in the field of environmental biology, can solve the problems of long detection cycle, long maturity time, and the instrument is not easy to carry.

Active Publication Date: 2021-01-29
SOUTH CHINA UNIV OF TECH +1
View PDF11 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, the detection cycle is long. Although green fluorescent protein has many advantages, it takes a long time to mature, and it usually takes 2 to 3 hours to fully mature, which is not conducive to rapid detection.
Furthermore, the quantification of GFP requires the use of expensive instruments, and the instruments are not easy to carry, which is not conducive to on-site real-time detection

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Whole-cell biosensor for detecting heavy metal ions in water-soluble samples and its construction and application
  • Whole-cell biosensor for detecting heavy metal ions in water-soluble samples and its construction and application
  • Whole-cell biosensor for detecting heavy metal ions in water-soluble samples and its construction and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 Obtaining of Heavy Metal Ion Response Recombinant Bacteria

[0085] Using pSB1C3 as the starting vector (iGEM, http: / / parts.igem.org / Part:pSB1C3), a heavy metal responsive vector was constructed. The schematic diagram of heavy metal responsive carrier construction is as follows: figure 1 shown. The specific construction method is as follows:

[0086] 1. Synthesize the response protein and bidirectional promoter sequence (SEQ ID No.1 and SEQID No.2) of mercury and lead ion response respectively, the sequence from 5' to 3' is EcoRI, NotI, XbaI, response protein reverse complement Encoding genes, bidirectional promoter, SpeI, NotI and PstI.

[0087] 2. Synthetic cleavage gene SRRz, its sequence is shown in SEQ ID No.3, and the sequence from 5' to 3' is EcoRI, NotI, XbaI, SRRz cleavage gene, SpeI, NotI and PstI.

[0088] 3. Synthetic terminator T rrnB , its sequence is shown in SEQ ID No.4, and the sequence from 5' to 3' is EcoRI, NotI, XbaI, T rrnB Terminat...

Embodiment 2

[0095] Example 2 Cell Lysis Efficiency Rapid Detection of Sample Heavy Metal Ion Concentration

[0096] 1. Recovery and activation of recombinant E. coli

[0097] The recombinant Escherichia coli was streaked on the LB plate medium from the -80°C refrigerator, and cultured at 37°C for 14h. A single colony was picked, inoculated into LB medium, and cultured at 37°C and 220rpm for 12-16h.

[0098] 2. Preparation of Escherichia coli detection solution

[0099] Inoculate the resuscitated and activated bacterial solution into fresh LB medium at a volume ratio of 1:50, and cultivate to OD at 35-37°C and 220rpm 600 When the temperature reaches 0.4~0.8, add 0.1mM IPTG to the Erlenmeyer flask, continue to cultivate for 0.5h, and obtain Escherichia coli detection solution.

[0100] 3. Contact with the test sample

[0101] The E. coli detection solution in the Erlenmeyer flask was divided into different test tubes, 5 mL in each test tube, 10 μL of the test sample was added into the t...

Embodiment 3

[0116] Example 3 Combined with the semi-quantitative rapid detection of heavy metal ion gel by chromogenic gel method

[0117] 1. Chromogenic gel preparation

[0118] 1.1 Add 0.4g agar powder to 20mL Z buffer, heat it in a microwave oven to dissolve and keep it warm in a 70°C water bath for later use.

[0119] 1.2 Add 40 mg / mL X-gal solution to the above solution to a final concentration of 1 mg / mL, mix well, pipette 200 μL into a 96-well plate, cool and solidify at room temperature, and store at 4°C for later use. It can be stored for more than 3 months.

[0120] 2. Draw standard curve of chromogenic gel

[0121] According to steps 1-3 of Example 2, the test bacteria were activated, cultured and contacted with the sample to be tested. Take 10 μL of the bacterial solution and add it to the well containing the detection gel, and incubate at 37°C for 30min. With different concentrations of metal ions, the gel will show different shades of blue ( Figure 4 ).

[0122] Use t...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a whole-cell biosensor for detecting heavy metal ions in a water-soluble sample and construction and application thereof. The operation object is escherichia coli, no pathogenic risk exists, and the operation is simple and easy to implement. An SRRz cracking gene is used as a report element, so that microbial bacteria are cracked, and the turbidity of bacterial liquid is obviously changed. Detection can be performed through a visible spectrophotometer, and a result can be obtained through direct observation with naked eyes. The response is quick, and only 30-60 minutesare needed from sample contact to result acquisition. X-gal is used for detecting beta-galactosidase released to the outside of the cells, and the operation is simple and convenient. Expensive instruments are not needed in application, so that the detection cost is greatly reduced, and field detection becomes possible. The extracellular beta-galactosidase activity is accurately quantified by usingchromogenic gel containing oNPG, the detection limit is low, and the detection result is accurate. Technical support is provided for daily heavy metal pollution detection and sudden heavy metal pollution detection.

Description

technical field [0001] The invention belongs to the field of environmental biotechnology, and in particular relates to a whole-cell biosensor for highly sensitive detection of heavy metal ions (divalent mercury ions or lead ions) in water-soluble samples and its construction and application. Background technique [0002] With the development of industry, heavy metal pollution has become increasingly serious and has become one of the prominent environmental problems. Heavy metals are highly toxic, easy to accumulate in organisms and difficult to degrade, and pose a huge threat to human health after entering the food chain. Therefore, the detection technology of heavy metal ions is particularly important. The detection techniques for heavy metal ions mainly include atomic absorption spectrometry, atomic fluorescence spectrometry, inductively coupled plasma, ultraviolet-visible spectrophotometry, high performance liquid chromatography, electrochemical analysis, biological detec...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/53C12Q1/10C12Q1/34C12R1/19
CPCC12Q1/10C12Q1/34G01N33/53
Inventor 卓敏李爽胡日荣吴柏华彭晓春
Owner SOUTH CHINA UNIV OF TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com