CN110456055A - A kind of biosensor and preparation method thereof detecting PS excretion body - Google Patents
A kind of biosensor and preparation method thereof detecting PS excretion body Download PDFInfo
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- CN110456055A CN110456055A CN201910746802.XA CN201910746802A CN110456055A CN 110456055 A CN110456055 A CN 110456055A CN 201910746802 A CN201910746802 A CN 201910746802A CN 110456055 A CN110456055 A CN 110456055A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57449—Specifically defined cancers of ovaries
Abstract
The present invention provides a kind of biosensors and preparation method thereof of phosphatidyl serine (PS) excretion body.The present invention is using PS specific recognition peptide as recognition component, and the bio-sensing electrode of identification peptide assembling is as capture substrate, by g-C3N4The electrogenerated chemiluminescence probe of nanometer sheet, luminol-AuNPs and identification peptide composition is as signal probe.With the PS excretion body in capture substrate and signal probe specific recognition sample, sandwich structure is formed, detection signal is generated by signal probe.PS identifies that peptide can be specific in conjunction with the PS on tumour source excretion body, and the PS identification peptide structure is simple, and affinity and selectivity are high, and not vulnerable to such environmental effects such as pH, temperature, stability is good.The present invention can be achieved highly sensitive and highly selective PS excretion physical examination and survey.
Description
Technical field
The invention belongs to excretion body detection technique fields, and in particular to a kind of to detect tumour source excretion body based on identification peptide
Electrochemiluminescsensor sensor and preparation method thereof.
Background technique
Cancer has become the number one killer for threatening human health, and early detection and accurate detection are to reduction mortality
It is of great practical significance with survival rate is improved.For a long time, tumor tissue biopsy is marked frequently as the gold of diagnosing tumor
Standard, but with going deep into tumor research, scientist's discovery organizes Biopsy to have one during the diagnosing and treating of cancer
Fixed limitation.It is mainly shown as that tumour has heterogeneity, certain patients are not suitable for doing tissue biopsy, organize the hysteresis quality of biopsy
It is unfavorable to the treatment of patient.Tumor-marker analyte detection is widely used in clinic as a kind of tumor diagnosis method,
But clinically used tumor markers are such as, and the sensitivity of alpha-fetoprotein (AFP) and carcinomebryonic antigen (CEA) etc. and early detective rate are all
It can not meet the demand that clinical early diagnosis is early controlled.Therefore, for the diagnosis of cancer and detection technique, more stringent requirements are proposed.
Excretion body, the extracellular vesica of nanoscale bilayer film property, diameter are 30-150 nm.Excretion body is entrained and transmits
The a large amount of informational molecule of mother cell, including lipid, protein and nucleic acid etc. form a kind of completely new cell-tocell transmitting
System influences the physiological status of cell and closely related with the generation of a variety of diseases and process, such as the excretion of tumor cell secretion
Body can enter the capillary in lymphatic system and tumor tissues, play the regulation work for the processes such as malignant tumour being occurred and being shifted
With.Therefore, excretion body is considered as the tumor markers of new generation of early diagnosis of cancer, and exploitation is used for highly sensitive and high specific
The analytical technology for detecting tumour excretion body is particularly significant.
So far, research of most of detection tumour source excretion bodies for cancer diagnosis is all concentrated on as specific tumors
In the excretion body protein matter and nucleic acid of type substitute marker.It recent studies have shown that, Healthy People, benign tumour and malignant tumour are suffered from
The excretion body of phosphatidyl serine (PS) positive has obviously difference in blood between person, to PS positive tumor in blood sample
The qualitative assessment of excretion body can detect the malignant tumour of very early stage before clinical evidence, it was demonstrated that the PS positive is swollen in blood
Tumor excretion body is a kind of general reliable potential marker of cancer early diagnosis.Therefore it realizes to PS positive tumor excretion body in blood sample
Accurately, efficient detection can not only be greatly reduced diagnosis cost, reduce the invasive surgical to patient, examine the early stage of cancer
Disconnected and curative effect evaluation also has very high clinical value.
Currently, exploitation for the physical examination of PS excretion survey method it is less, mainly include Western blotting, flow cytometry and
Enzyme linked immunosorbent assay.And these methods have certain disadvantages, such as expensive equipment, complicated for operation and time-consuming.In addition, above-mentioned
Though immune combine of common antigen-antibody can obtain higher detection sensitivity in PS excretion body detecting method, screening is special
Property the antibody that combines often step is complicated, but when antibody uses specific position combine it is difficult, on the one hand, protein identification element
The synthesis of (antibody, receptor protein etc.) and purification process very complicated lack steady under various testing conditions or when long term storage
It is qualitative, it is denaturalized vulnerable to such environmental effects such as pH, temperature, and price is costly.
Summary of the invention
The purpose of the present invention is to provide a kind of detection PS positive tumor source excretion body biosensor and preparation methods, lead to
It crosses identification peptidyl probe to combine the high specific of PS positive excretion body, realizes and the PS positive excretion body in sample is carried out fastly
Fast, highly sensitive detection, solve existing excretion physical examination survey in it is complicated for operation, sensitivity is low, common recognition component (antibody, nucleic acid
Aptamers etc.) it synthesizes with purification process very complicated, deficient in stability under various testing conditions or when long term storage, vulnerable to
The such environmental effects such as pH, temperature and be denaturalized, it is expensive the problems such as.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
It is a kind of detect phosphatidyl serine PS positive excretion body biosensor, the sensor using PS specific recognition peptide as
Recognition component, the bio-sensing electrode of identification peptide assembling is as capture substrate, by g-C3N4Nanometer sheet, luminol-AuNPs and knowledge
The electrogenerated chemiluminescence probe of other peptide composition is as signal probe.
A kind of PS specific recognition peptide, amino acid sequence areFNFRLKAGAKIRFGRGC, underscore part is in sequence
PS unique identification sequence, RGC are key Lian Xulie, and the sulfydryl in terminal cysteine C can be combined with gold.
The preparation method of the capture substrate, comprising the following steps: using chlorauric acid solution as electro-deposition bottom liquid, glass-carbon electrode
The glass-carbon electrode of AuNPs modification is prepared using potentiostatic electrodeposition method for working electrode, further rinsed with deionized water,
After room temperature is dried, modified electrode is immersed in identification peptide solution and is incubated for and is rinsed;Modified electrode is then immersed into ox blood again
It is closed in albumin, obtains the bio-sensing electrode of identification peptide assembling.
The preparation method of the signal probe, comprising the following steps: by g-C3N4Nanometer sheet and luminol-AuNPs mixing are equal
After even, then stirring is centrifuged 30 minutes removing supernatants with 6000 rpm and obtains luminol-AuNPs@g-C3N4Sediment will obtain
Luminol-AuNPs@g-C3N4Sediment and identification peptide form Au-S key by sulfydryl and golden coordination and are combined
Obtain luminol-AuNPs@g-C3N4/ identification peptide signal probe.
A kind of test method detecting PS excretion body biosensor, comprising: by excretion liquid solution to be measured in the life
It is incubated on object sensing electrode, makes the capture of PS positive excretion body on a biosensor electrode, then by the luminol-
AuNPs@g-C3N4/ identification peptide signal probe solution is incubated on the biological sensor electrode that capture has PS positive excretion body, makes to visit
Needle is incorporated on the PS positive excretion body of biological sensor electrode, to form signal probe and biological sensor electrode folder load PS
The identification peptidyl sandwich type biosensor of excretion body is containing H to the electroluminescent chemiluminescence biosensor2O2PBS solution
In detected, by signal probe generate electrochemiluminescence signal, to indicate the content of PS excretion body.
Compared with prior art, the beneficial effects of the present invention are:
The present invention detects PS excretion body with combining identification peptide technology to construct efficient electrogenerated chemiluminescence (ECL) sensing platform.
Antibody is replaced using specific recognition peptide in the strategy, tumour source excretion body and normal excretion body can be effectively distinguished, improve inspection
The selectivity and stability of examining system.In addition, the sensor can be used for detecting the tumour source excretion body in serum, it is suitable for clinic
Detection.
Detailed description of the invention
Fig. 1 is the assembling and detection schematic diagram of disclosure Electrochemiluminescsensor sensor.
Fig. 2 is luminol-AuNPs@g-C prepared by embodiment 13N4The transmission electron microscope figure of nano material.
Fig. 3 is the influence (A) and calibration curve (B) that various concentration PS excretion body responds ECL in the preparation of embodiment 1.
Fig. 4 is the ECL that SKOV3ip ovarian tumor cell and HMrSV5 normal cell secrete excretion body in the preparation of embodiment 4
It responds (A) and detects the SKOV3ip excretion body (B) in different medium.
Specific embodiment
The present invention is further illustrated with specific embodiment with reference to the accompanying drawing, but the present invention is not limited to following
Embodiment.
As background technique is introduced, need to develop a kind of for highly sensitive and specific detection PS excretion body analysis
Technology, in view of this, this patent provides a kind of detection PS positive tumor source excretion body biosensor and preparation method.
The assembling of Electrochemiluminescsensor sensor and experimental principle:
Assembling and the testing principle of Electrochemiluminescsensor sensor as shown in Figure 1, deposit AuNPs on the glass-carbon electrode surface (GCE),
AuNPs has excellent biocompatibility and electric conductivity, and can provide more recognition sites to fix PS identification peptide, thus
More excretion bodies are captured, and bovine serum albumin(BSA) is used to prevent non-specific adsorption as sealer.Then luminol-is synthesized
AuNPs@g-C3N4/ PS identifies that peptide signal probe in conjunction with excretion body, forms sandwich structure, finally utilizes g-C3N4And AuNPs
To H2O2Dual catalytic performance, improve luminol-H2O2The ECL luminous efficiency of system is believed using the electrogenerated chemiluminescence of generation
Number power come reflect capture PS positive excretion body number, thus achieve the purpose that detect excretion body.Electrogenerated chemiluminescence letter
It is number relatively strong then in the luminol-AuNPs@g-C of electrode surface3N4/ PS identifies that peptide signal probe is more, to indirectly illustrate
PS positive excretion body on electrode is more.
In order to enable those skilled in the art can clearly understand the technical solution of the disclosure, below with reference to tool
The technical solution of the disclosure is described in detail in the embodiment of body.
Embodiment 1
1、g-C3N4The synthesis of nanometer sheet and luminol-AuNPs
G-C is prepared by being directly pyrolyzed melamine3N4.Melamine heats 4 hours at 550 DEG C, keeps 4 again at this temperature
Hour is simultaneously cooled to room temperature.Then the yellow product obtained is blocky g-C3N4Powder.Following synthesis g-C3N4Nanometer sheet: to 1 g
Ontology g-C3N4100 mL 5mol/L HNO are added in powder3, flow back 24 hours at 125 DEG C, and cool down at room temperature.It will be white
Color product is centrifuged with 12000 rpm, with milli-Q water to close to neutral pH, and is redispersed in water.In order to obtain
Gained suspension is ultrasonically treated 4 hours by the nanometer sheet of even size, then remaining to remove with 8000 rpm centrifugation 30 minutes
Unpeeled g-C3N4Nano particle and large-area nano piece.Then, the supernatant of acquisition is centrifuged with 5000 rpm outstanding to remove
Supernatant liquid.Finally, product to be dried to 12 hours in 35 DEG C of vacuum drying oven to obtain g-C3N4Nanometer sheet is spare.Luminol is molten
Solution is made into the solution of 15 mM in NaOH solution, and the 2.4 mL solution and the gold chloride of 0.25 mM of 20 mL is then taken to mix,
30 min are stirred at room temperature.It is added dropwise under violent stirring into mixed solution, 0.1 M newly configured with cold water
NaBH4, solution becomes bluish violet from faint yellow, continues to stir 20 min can be obtained luminol-AuNPs solution.
2, luminol-AuNPs@g-C3N4The synthesis of/PS identification peptide probes
By 50 mg g-C3N4Nanometer sheet and 50 mL luminol-AuNPs solution after mixing, stir 24 h, then at room temperature
30 minutes removing supernatants, which are centrifuged, with 6000 rpm obtains luminol-AuNPs@g-C3N4Sediment, Fig. 2 are luminol-AuNPs@g-
C3N4The transmission electron microscope figure of nano material.Luminol-AuNPs@the g-C that will be obtained3N4Sediment and identification peptide pass through mercapto
Being combined 10 h at base and golden 4 DEG C of coordination can be obtained luminol-AuNPs@g-C3N4/ PS identifies peptide signal probe.
3, the assembling of biosensor
It is reaction electrolyte with 1% chlorauric acid solution of quality, is arrived AuNPs modification by 10 s of potentiostatic electrodeposition at -0.2 V
On clean glass-carbon electrode, flushing is dried;Then modified electrode 4 DEG C of 0.5 h of incubation in 0.2 μM of identification peptide solution are immersed to incubate
It educates;Then modified electrode is immersed in 0.2% bovine serum albumen solution again and is incubated for 0.5 h at 4 DEG C, is obtained after cleaning drying.
The PS positive SKOV3ip excretion body of various concentration is incubated for 1 h in capture substrate at room temperature.It is obtained after cleaning drying
Peptide/deposition gold/GCE is identified to PS positive excretion body/bovine serum albumin/PS.
To capture after the electrode of excretion body cleans drying with distilled water, by probe solution on above-mentioned electrode at room temperature under
It is incubated for 1 h, to be cleaned after fully reacting with distilled water, is dried with nitrogen, the electrogenerated chemiluminescence biology prepared
Sensor.
4, the detection of PS excretion body
Excretion body is analyzed using this electroluminescent chemiluminescence biosensor.In the case where scanning current potential is 0.2-0.8 V, in light
Electric multiplier tube is to contain 10 mM H to the electroluminescent chemiluminescence biosensor under 800 V2O20.1 pH=7.4 M PBS
Middle carry out electrochemiluminescdetection detection obtains the ECL intensity of the excretion body standard solution of various concentration, further according to excretion body standard
The concentration of solution and corresponding ECL intensity draw linear relationship curve.Fig. 3 is the shadow that various concentration excretion body responds ECL
(A) and calibration curve (B) are rung, as seen from the figure, with the increase of excretion bulk concentration, electrochemiluminescence signal is gradually increased.In
The concentration of excretion body is 102-107Within the scope of a/μ L, the logarithm of the size and the concentration of excretion body of electrochemiluminescence signal is in
Linear relationship, detection are limited to 39/μ L.The ECL intensity that excretion liquid solution to be measured is measured according to the above method, further according to described
Linear relationship curve obtains the concentration of PS positive tumor source excretion body in excretion liquid solution to be measured.
Embodiment 2
1, luminol-AuNPs@g-C3N4The synthesis of/PS identification peptide probes
By 100 mg g-C3N4Nanometer sheet and 50 mL luminol-AuNPs solution after mixing, stir 18 h, so at room temperature
30 minutes removing supernatants, which are centrifuged, with 6000 rpm afterwards obtains luminol-AuNPs@g-C3N4Sediment, Fig. 2 are luminol-AuNPs@
g-C3N4The transmission electron microscope figure of nano material.Luminol-AuNPs@the g-C that will be obtained3N4Sediment passes through with identification peptide
Being combined 6 h at sulfydryl and golden 30 DEG C of coordination can be obtained luminol-AuNPs@g-C3N4/ PS identifies peptide signal probe.
2, the assembling of biosensor
It is 1% chlorauric acid solution with mass fraction is reaction electrolyte, by 30 s of potentiostatic electrodeposition by AuNPs at -0.2 V
It modifies on glass-carbon electrode, flushing is dried;Then modified electrode 4 DEG C of 2 h of incubation in 0.5 μM of identification peptide solution are immersed to incubate
It educates;Then modified electrode is immersed in 1% bovine serum albumen solution again and is incubated for 1 h at 4 DEG C, is obtained after cleaning drying.
The PS positive SKOV3ip excretion body of various concentration is incubated for 1 h upper at room temperature.The PS positive is obtained after cleaning drying
Excretion body/bovine serum albumin/PS identifies peptide/deposition gold/GCE.
To capture after the electrode of excretion body cleans drying with distilled water, by probe solution on above-mentioned electrode at room temperature under
It is incubated for 1 h, to be cleaned after fully reacting with distilled water, is dried with nitrogen, the electrogenerated chemiluminescence biology prepared
Sensor.
Embodiment 3
1, luminol-AuNPs@g-C3N4The synthesis of/PS identification peptide probes
By 50 mg g-C3N4Nanometer sheet and 100 mL luminol-AuNPs solution after mixing, stir 36 h, so at room temperature
30 minutes removing supernatants, which are centrifuged, with 6000 rpm afterwards obtains luminol-AuNPs@g-C3N4Sediment, Fig. 2 are luminol-AuNPs@
g-C3N4The transmission electron microscope figure of nano material.Luminol-AuNPs@the g-C that will be obtained3N4Sediment passes through with identification peptide
Being combined 8 h at sulfydryl and golden 40 DEG C of coordination can be obtained luminol-AuNPs@g-C3N4/ PS identifies peptide signal probe.
2, the assembling of biosensor
Using 1% chlorauric acid solution of quality as electro-deposition bottom liquid, AuNPs modification is arrived by 20 s of potentiostatic electrodeposition at -0.2 V
On glass-carbon electrode, flushing is dried;Then modified electrode 4 DEG C of 12 h of incubation in 1 μM of identification peptide solution are immersed to be incubated for;Then again
Modified electrode is immersed in 0.2% bovine serum albumen solution and is incubated for 0.5 h at 4 DEG C, is obtained after cleaning drying.
The PS positive SKOV3ip excretion body of various concentration is incubated for 1 h upper at room temperature.The PS positive is obtained after cleaning drying
Excretion body/bovine serum albumin/PS identifies peptide/deposition gold/GCE.
To capture after the electrode of excretion body cleans drying with distilled water, by probe solution on above-mentioned electrode at room temperature under
It is incubated for 1 h, to be cleaned after fully reacting with distilled water, is dried with nitrogen, the electrogenerated chemiluminescence biology prepared
Sensor.
Embodiment 4
The selectivity and actual sample of biosensor detect
Since there are notable differences for content on normal excretion body and tumour source excretion body for phosphatidyl serine, using this
The PS positive excretion body of sensor platform tumor cell secretion.The application has distinguished normal cell HMrSV5 using this method
The excretion body secreted with ovarian cancer cell SKOV3 ip, and the PS positive excretion body in practical serum is detected.Fig. 4 is
To the SKOV3ip excretion body in the ECL response (A) and detection different medium of SKOV3ip and HMrSV5 cell secretion excretion body
(B).This electroluminescent chemiluminescence biosensor divides the ovarian tumor cell SKOV3ip and normal cell HMrSV5 of same concentrations
The excretion body secreted is detected, and as a result has apparent difference, and it is good to illustrate that the peptide based sensor has tumour source excretion body
Selectivity.In order to assess the clinical application of sensor detection tumour source excretion body, different medium, i.e. phosphoric acid are detected respectively
Salt buffer solution (PBS) and 5% go same concentrations in the cow's serum (FBS) of excretion body PS positive excretion body, as a result basic one
It causes, shows that this method can perform well in complicated biological sample.
Above embodiment described details of the invention, but the present invention is not limited thereto.In skill of the invention
In art conception range, to any improvement and a variety of simple deformations that technical solution of the present invention carries out, guarantor of the invention is belonged to
Protect range.
Claims (7)
1. a kind of biosensor of the positive excretion body of detection phosphatidyl serine (PS), feature exist: with PS specific recognition
Peptide identifies the bio-sensing electrode of peptide assembling as capture substrate, by g-C as recognition component3N4Nanometer sheet, luminol-
The electrogenerated chemiluminescence probe of AuNPs and identification peptide composition is as signal probe.
2. PS specific recognition peptide according to claim 1, it is characterised in that: amino acid sequence isFNFRLKAGAKIRFGRGC, underscore part is PS unique identification sequence in sequence, and RGC is key Lian Xulie, half Guang ammonia of end
Sulfydryl in sour C can be combined with gold.
3. capture substrate according to claim 1, preparation method are characterized in that: using chlorauric acid solution as electro-deposition bottom
Liquid, glass-carbon electrode are working electrode, using potentiostatic electrodeposition method, prepare the glass-carbon electrode of AuNPs modification, further spend
After ionized water rinses, room temperature is dried, modified electrode is immersed in identification peptide solution and is incubated for and is rinsed;It then again will modification electricity
Pole, which is immersed in bovine serum albumin, to be closed, and the bio-sensing electrode of identification peptide assembling is obtained.
4. in capture substrate preparation method according to claim 3, the mass fraction of the chlorauric acid solution is 1%, permanent electricity
The voltage of position deposition is -0.2 V, and sedimentation time is 10~30 s;The concentration of the identification peptide aqueous solution is 0.2~1 μM, In
4 DEG C of 0.5~12 h of incubation;The mass fraction of bovine serum albumin aqueous solution is 0.2%~1%, and 0.5~1 h is incubated at 4 DEG C.
5. signal probe according to claim 1, preparation method are characterized in that: by g-C3N4Nanometer sheet and luminol-
After mixing, then stirring is centrifuged 30 minutes removing supernatants with 6000 rpm and obtains luminol-AuNPs@g-C AuNPs3N4It is heavy
Starch, the luminol-AuNPs@g-C that will be obtained3N4Sediment and identification peptide by sulfydryl and golden coordination formed Au-S key into
Row, which combines, can be obtained luminol-AuNPs@g-C3N4/ identification peptide signal probe.
6. in signal probe preparation method according to claim 5, the g-C3N4Nanometer sheet and luminol-AuNPs solution
Ingredient proportion be (0.5~1) mg:(0.5~10) mL;18~36 h are stirred at room temperature;Luminol-AuNPs@the g-
C3N4Sediment and identification peptide combination temperature are 4~40 DEG C, and the time is 6~10 h.
7. a kind of test method for detecting PS excretion body biosensor, characterized in that want excretion liquid solution to be measured in right
It is incubated on bio-sensing electrode described in asking 1 or 3, makes the capture of PS positive excretion body on a biosensor electrode, it then will power
Benefit require 1 or 5 described in luminol-AuNPs@g-C3N4/ identification peptide signal probe solution has the life of PS positive excretion body in capture
It is incubated on object sensor electrode, is incorporated in probe on the PS positive excretion body of biological sensor electrode, to form signal spy
Needle and biological sensor electrode folder carry the identification peptidyl sandwich type biosensor of PS excretion body, raw to the electrogenerated chemiluminescence
Object sensor is in (the H containing hydrogen peroxide2O2) phosphate buffer solution (PBS) in detected, generated by signal probe electroluminescent
Chemiluminescence signal, to indicate the content of PS excretion body.
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