CN108362752A - A kind of whole-cell biological electrochemical sensor of detection trimethylamine oxide - Google Patents
A kind of whole-cell biological electrochemical sensor of detection trimethylamine oxide Download PDFInfo
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Abstract
A kind of whole-cell biological electrochemical sensor of detection trimethylamine oxide, the analysis that can be used for trimethylamine oxide are quantitative.The present invention uses three-electrode system, and the reaction strain detected as experimental analysis using electricity production bacterium Shewanella MR 1, is applied to trimethylamine oxide（TMAO）Specificity detection.This detecting system is using the mixed liquor of the Shewanella liquid and buffer solution cultivated after activation as the electrode reaction buffer solution of whole-cell biological sensor detecting system, reaction solution is added with the trimethylamine oxide sample of certain concentration, detect and recording device for trimethylamine oxide kinetic current change curve, according to the relationship that curent change curve and trimethylamine oxide concentration change, the concentration of the trimethylamine oxide in sample is calculated.This detecting system controllability is strong, and demand for labour amount is small, affected by environment small, reduces cost of determination and operation difficulty, and highly efficient easily survey tool is provided for the detection of the trimethylamine oxide of marine product Quality Detection etc..
Description
Technical field
The present invention relates to a kind of whole-cell biological electrochemical sensors of detection trimethylamine oxide, belong to biochemistry detection technology
Field.
Background technology
Trimethylamine oxide（Trimetlylamine oxide, TMAO）It is widely present in aquatic/marine animals body, is a kind of heavy
The nontoxic mesostate wanted is commonly used for mariculture industry, animal husbandry, and major function, which has, promotes muscle cell proliferation to promote
Increase into musculature, increase the works such as bile volume inhibition fat deposition, the osmotic pressure of balance aquatic animal, stable protein structure
With, and there is attractant effect as the additive of feed because of its special delicious sweet taste.Additionally while trimethylamine oxide is
The main component of seafood taste, but its trimethylamine for resolving under certain condition and dimethylamine be fish corruption stench at
Point, therefore for the monitoring of trimethylamine oxide for ensureing that it is huge that safe and healthy and feedstuff industry the development of seafood has
Meaning.
It is reported that（Wang Wei accounts for, Tian Fengshi etc., trimethylamine oxide and relationship between atherosclerosis progress [R] .43-4,
2015.）Trimethylamine oxide has certain relevance with atherosclerosis, detects to the quick quantization of trimethylamine oxide and is examined in disease
Disconnected aspect is also significant.
The existing detection technique to trimethylamine oxide common are high performance liquid chromatography, capillary zone electrophoresis skill
Art, mass spectrography, capillary isotachophoresis technology etc..But above method generally existing needs to carry out complicated sample preprocessing
（Solid phase extractions object or derivative）, equipment is expensive or the associated problem of physico-chemical process such as low specificity.
Biosensor is a kind of sensitive to biological substance and converts its concentration to the instrument that electric signal is detected, and is somebody's turn to do
Class system has application because of its efficient, convenient, high specific advantage in terms of very much.
Three-electrode system refers to by working electrode, reference electrode and the electrode system formed to electrode.The system can be used
In the conversion element of bioelectrochemical sensor, biochemical signals are converted to the electric signal conducive to detection.
Invention content
The purpose of the present invention be exactly for the above-mentioned prior art in the presence of shortcoming and a kind of detection is provided and is aoxidized
The whole-cell biological electrochemical sensor of trimethylamine quantization detects trimethylamine oxide for specificity.The present invention is electrochemical student
Object Sensor Design opens New Century Planned Textbook, and for marine product Quality Detection in terms of trimethylamine oxide detection provide it is a kind of high
Effect, smaller survey tool convenient, affected by environment.
The purpose of the present invention can be realized by following technique measures：
The whole-cell biological electrochemical sensor of the detection trimethylamine oxide of the present invention is through the following steps that be prepared：
（1）The Shewanella inoculation of ultralow temperature preservation is subjected to actication of culture, obtains the thalline of activation；
（2）By 2%-15%（Volume ratio compared with electrode reaction buffer solution）Activation thalline centrifugation, it is slow that electrode reaction is added
Fliud flushing obtains mixing liquid；
（3）By step（2）The mixing liquid of gained is added by working electrode, the three-electrode system assembled to electrode and reference electrode
In, then connection signal detecting system, collectively constitutes whole-cell biological electrochemical sensor；
（4）Using electrochemical workstation or analog instrument as signal detection system, connects above-mentioned whole-cell biological electrochemistry and pass
The three electrode decorum of sensor, and external voltage is controlled, record current output signal, after electric current output up to after stablizing, successively to complete thin
Sample is added in born of the same parents' bioelectrochemical sensor, and records the change curve of current signal in reactor, this curve is computed can
Obtain the trimethylamine oxide concentration of counter sample..
Step of the present invention（1）Described in Shewanella beShewanella oneidensisMR-1 strains.
Step of the present invention（2）Described in electrode reaction buffer solution be to be prepared by following substance：M9 culture mediums：
Na2HPO4·12H2O 17.8 g/L、KH2PO4 3 g/L、 NaCl 0.5 g/L、NH4Cl 10.5 g/L ；LB Liquid Cultures
Base：10 g/L of tryptone, 5 g/L of yeast extract, 5 g/L of sodium chloride（pH=7.0）；The sodium lactate of 18 mM, 0.1 mM
CaCl2With the MgSO of 1 mM4。
Step of the present invention（3）Described in working electrode be carbon cloth electrode；The reference electrode be saturated calomel electrode or
Silver/silver chloride electrode；Described is platinum electrode to electrode.
Step of the present invention（4）Described in electrochemical workstation or analog instrument need by controlling potential device and note
Record the device composition of electric current output., such as electrochemical workstation CHI660E（Shanghai Chen Hua Instrument Ltd.）；Step（4）In
The full cell electrochemica biological sensor load is -1.5~1 volt in the external voltage of working electrode.
Step in the present invention（1）The Shewanella（Shewanella oneidensis ）The actication of culture of MR-1 can
Think Liquid Culture or solid culture：For Liquid Culture when, activated bacterium solution is centrifuged, is abandoned bacterial sediment after supernatant again
It is suspended in fresh fluid nutrient medium；For solid culture when, scrape bacterium colony, be suspended in fresh culture.
In addition, it is used in the present invention by three-electrode system structure from biosensor, be using Shewanella as
The identifying system of detectable substance trimethylamine oxide, in the periplasmic space of Shewanella, there are the oxidations that catalysis oxidation trimethylamine restores
Trimethylamine reductase, in solution trimethylamine oxide reductase reduction-oxidation trimethylamine and by the electronics of generation by Shewanella
Electron transport system is transmitted to external circuit and forms the fluctuation of current signal, reduction reaction be formed by electronics and current strength at
Direct ratio, therefore the current signal detected can form correlation with the additive amount of trimethylamine oxide.
Beneficial effects of the present invention are as follows：
（1）Biosensor provided by the invention can detect the electron transmission that trimethylamine oxide is reacted in vivo, with this
Detection architecture is constituted, there is specificity to the detection of trimethylamine oxide；
（2）According to simple electrochemical apparatus to realize that voltage controls the record with current signal, testing cost is cheap；
（3）The amount of labour is low, and labour demand is few.
Description of the drawings
Fig. 1 is the bioelectrochemical sensor schematic diagram of the present invention.
Fig. 2 is the standard curve measured by detection trimethylamine oxide biosensor provided by the invention.
Fig. 3 is the specific test of sensor.
Specific implementation mode
The present invention is further described below with reference to embodiment：
Heretofore described Shewanella（Shewanella oneidensis）Purchased from American Type Culture collection
(ATCC) ；The Shewanella（Shewanella oneidensis ）The activation culture mode of MR-1 is for Liquid Culture or admittedly
Body culture；When activation culture is Liquid Culture, activated bacterium solution is centrifuged, is resuspended in bacterial sediment newly after abandoning supernatant
In fresh fluid nutrient medium；When activation culture is solid culture, bacterium colony is scraped, is suspended in fresh culture.It is no longer superfluous below
It states.
Embodiment 1：
（1）The Shewanella inoculation of ultralow temperature preservation is subjected to actication of culture, obtains the thalline of activation；
（2）By 15 mL（Volume ratio compared with electrode reaction buffer solution is 15%）The thalline of activation centrifuges, and electrode reaction is added
Buffer solution obtains mixing liquid；Steps are as follows for the preparation method of the electrode reaction buffer solution：
The configuration of electrode reaction buffer solution：By 95 mL M9 culture mediums（Containing Na2HPO4·12H2O 17.8 g/L、KH2PO4 3
g /L、NaCl 0.5 g/L、NH4Cl 1 g/L）With 5 mL LB fluid nutrient mediums（It is extracted containing 10 g/L of tryptone, yeast
5 g/L of 5 g/L of object and sodium chloride, pH=7.0）It is sufficiently mixed, in addition adds sodium lactate, calcium chloride, magnesium sulfate and keeps it dense eventually
Degree respectively reaches 18 mM, 0.1 mM and 1 mM；Reaction solution needs 121 degree of 20 min autoclaves sterilizing postcooling spare；
（3）The assembling of whole-cell biological electrochemical sensor：Using the working method of three-electrode system, with the 1 × 2 of the connection of titanium silk
Cm carbon cloths using saturated calomel electrode as reference electrode, and use electricity as working electrode using platinum electrode as to electrode
Chem workstation CHI660e（Shanghai Chen Hua Instrument Ltd.）Control applies -0.6 volt of external voltage in working electrode.
（As shown in Fig. 1）
（4）The measurement of standard curve：Trimethylamine oxide standard solution is prepared, final concentration is respectively 1 mM, 0.5 mM（2-1
mM）, 0.25 mM（2-2 mM）, 0.125 mM（2-3 mM）, 0.0625 mM（2-4mM）, 0.03125 mM（2-5mM）,
0.01563 mM（2-6 mM）, 0.00781 mM（2-7mM）；By step（3）The whole-cell biological electrochemical sensor of making takes out
Afterwards, connection electrochemical workstation electric current to be output reaches stable, and the trimethylamine oxide standard solution of various concentration is added successively
Into bioelectrochemical sensor, the change curve of record current（See attached drawing 3）.By integral and calculating, various concentration is obtained
Trimethylamine oxide solution with its detect the equation of linear regression that is fitted to of peak area of the current-responsive curve of gained be y=
0.0065x+0.0004, the two are positively correlated, coefficient R2Reach 0.9988,（See attached drawing 2）.
Embodiment 2
The step of the present embodiment is with embodiment 1（1）-（3）It is identical, in step（4）In, prepare trimethylamine oxide, glucose, lemon
Different solution is added in bioelectrochemical sensor respectively for the standard solution of the identical gradient of acid, record detection gained
Current output signal, the results showed that, only trimethylamine oxide generates apparent current output signal（As shown in Figure 3）.
Embodiment 3
The present embodiment and the step in embodiment 1（1）~（3）Identical, compound concentration is respectively 0.3 mM, 0.4 mM, 0.5 mM
Standard solution, in step（4）It is middle that this three kinds of concentration are separately added into bioelectrochemical sensor, daily in triplicate, note
Record detection gained voltage output curve, so repeats 6 days, the results are shown in Table 1.
The stability test of table 1 TMAO detections（n=6）
| TMAO sample concentrations (mM) | Measured value（Mean value, mM） | The coefficient of variation (%) | The rate of recovery (%) |
| 0.3 | 0.287 | 2.33 | 95.66 |
| 0.4 | 0.41 | 1.6 | 102.61 |
| 0.5 | 0.505 | 4.61 | 101.01 |
According to table 1 it is found that when sample concentration is 0.3 mM, 0.4 mM and 0.5 tri- kinds of concentration of mM, the variation lines of 6 days measured values
The equal ＜ 5% of number, the rate of recovery reaches 95.66%-102.61%, illustrates that sensor has preferably the measurement of TMAO in the long period
Stability..
Claims (7)
1. a kind of whole-cell biological electrochemical sensor of detection trimethylamine oxide, it is characterised in that：The sensor is to pass through
Following steps are prepared：
（1）The Shewanella MR-1 inoculations of ultralow temperature preservation are subjected to actication of culture, obtain the thalline of activation；
（2）The thalline of activation is centrifuged, electrode reaction buffer solution is added, obtains mixing liquid；The thalline quantity of the activation is
The 2%-15% of electrode reaction buffer solution volume；
（3）By step（2）The mixing liquid of gained is added by working electrode, the three-electrode system assembled to electrode and reference electrode
In, then connection signal detecting system, forms whole-cell biological electrochemical sensor；
（4）Using electrochemical workstation or analog instrument as signal detection system, connects above-mentioned whole-cell biological electrochemistry and pass
The three electrode decorum of sensor, and external voltage is controlled, record current output signal, after electric current output up to after stablizing, successively to complete thin
Sample is added in born of the same parents' bioelectrochemical sensor, and records the change curve of current signal in reactor, this curve is computed can
Obtain the trimethylamine oxide concentration of counter sample.
2. the whole-cell biological electrochemical sensor of detection trimethylamine oxide according to claim 1, it is characterised in that：Step
Suddenly（1）Described in Shewanella beShewanella oneidensis MR-1 strains.
3. the whole-cell biological electrochemical sensor of detection trimethylamine oxide according to claim 1, it is characterised in that：Step
Suddenly（2）Described in electrode reaction buffer solution be to be prepared by following raw materials：M9 culture mediums：Na2HPO4·12H2O 17.8
g/L、KH2PO4 3 g/L、 NaCl 0.5 g/L、NH4Cl 10.5 g/L ；LB fluid nutrient mediums：10 g/L of tryptone, ferment
5 g/L of sodium chloride of female extract 5 g/L, pH=7.0；The CaCl of the sodium lactate of 18 mM, 0.1 mM2With the MgSO of 1 mM4。
4. the whole-cell biological electrochemical sensor of detection trimethylamine oxide according to claim 1, it is characterised in that：Step
Suddenly（3）Described in working electrode be carbon cloth electrode；The reference electrode is saturated calomel electrode or silver/silver chloride electrode；Institute
It is platinum electrode to state to electrode.
5. according to the whole-cell biological electrochemical sensor of the detection trimethylamine oxide described in claim 1, it is characterised in that：
Step（4）Described in electrochemical workstation or analog instrument need by controlling potential device and record current export device
Composition.
6. the whole-cell biological electrochemical sensor of detection trimethylamine oxide according to claim 1, it is characterised in that：
Step（4）Described in electrochemica biological sensor load working electrode external voltage be -1.5~1 volt.
7. the whole-cell biological electrochemical sensor of detection trimethylamine oxide according to claim 1, it is characterised in that：
Step（1）Described in the actication of culture of Shewanella MR-1 can be Liquid Culture or solid culture：For Liquid Culture when, will live
Bacterial sediment, is resuspended in after abandoning supernatant in fresh fluid nutrient medium by the bacterium solution centrifugation changed；For solid culture when, scrape
Bacterium colony is taken, is suspended in fresh culture.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113234623A (en) * | 2021-04-30 | 2021-08-10 | 福建农林大学 | Electroactive bacteria whole-cell moisture power generation device and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120040383A1 (en) * | 2010-08-12 | 2012-02-16 | Wei Jia | Methods and Kits Relating To Metabolite Biomarkers For Colorectal Cancer |
| JP2012112785A (en) * | 2010-11-24 | 2012-06-14 | Tohoku Univ | Evaluation method of dialysis membrane |
| CN104165909A (en) * | 2014-07-14 | 2014-11-26 | 江苏大学 | Biological electrochemical detection method of fumaric acid |
| CN105355929A (en) * | 2015-11-28 | 2016-02-24 | 成都九十度工业产品设计有限公司 | Microbial fuel cell based on trimethylamine oxide medium |
-
2018
- 2018-02-11 CN CN201810140062.0A patent/CN108362752A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120040383A1 (en) * | 2010-08-12 | 2012-02-16 | Wei Jia | Methods and Kits Relating To Metabolite Biomarkers For Colorectal Cancer |
| JP2012112785A (en) * | 2010-11-24 | 2012-06-14 | Tohoku Univ | Evaluation method of dialysis membrane |
| CN104165909A (en) * | 2014-07-14 | 2014-11-26 | 江苏大学 | Biological electrochemical detection method of fumaric acid |
| CN105355929A (en) * | 2015-11-28 | 2016-02-24 | 成都九十度工业产品设计有限公司 | Microbial fuel cell based on trimethylamine oxide medium |
Non-Patent Citations (1)
| Title |
|---|
| ELIZABETH A. WEST等: "Engineering a Native Inducible Expression System in Shewanella oneidensis to Control Extracellular Electron Transfer", 《ACS SYNTH. BIOL.》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113234623A (en) * | 2021-04-30 | 2021-08-10 | 福建农林大学 | Electroactive bacteria whole-cell moisture power generation device and application thereof |
| CN113234623B (en) * | 2021-04-30 | 2023-09-05 | 福建农林大学 | Electroactive strain whole-cell moisture power generation device and application thereof |
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Application publication date: 20180803 |