CN114088675A - Immunoliposome wrapping fluorescent dye and application - Google Patents

Immunoliposome wrapping fluorescent dye and application Download PDF

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CN114088675A
CN114088675A CN202111361157.3A CN202111361157A CN114088675A CN 114088675 A CN114088675 A CN 114088675A CN 202111361157 A CN202111361157 A CN 202111361157A CN 114088675 A CN114088675 A CN 114088675A
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solution
immunoliposome
fluorescent dye
liposome
patulin
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宋新杰
吴元锋
吴丽
孙娟
张尧
吕天凤
王天荣
王络
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Zhejiang Lover Health Science and Technology Development Co Ltd
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Zhejiang Lover Health Science and Technology Development Co Ltd
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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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching

Abstract

The invention discloses an immunoliposome wrapping fluorescent dye and application thereof, wherein the preparation method of the immunoliposome comprises the following steps: 1) dissolving DPPE and SATA in a chloroform solution, and carrying out ultrasonic treatment to form DPPE-SATA; dissolving DPPE-SATA, DPPC, DPPG and cholesterol in a mixed solution of chloroform and methanol, and carrying out ultrasonic treatment to form liposome; the liposome is encapsulated by the fluorescent dye to obtain the fluorescent dye encapsulated liposome; 2) modifying the liposome encapsulated by the fluorescent dye by using the antibody derived from the sulfo-KMUS to obtain the immune liposome encapsulating the fluorescent dye. The invention also discloses application of the immunoliposome coated with the fluorescent dye in fluorescent detection of patulin. The detection method using liposome wrapped in SRB as the identification and signal unit shows high sensitivity and specificity, and can be used for rapid detection of the clavulanic toxin.

Description

Immunoliposome wrapping fluorescent dye and application
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an immunoliposome wrapped with a fluorescent dye and an application thereof, in particular to a fluorescence detection technology for patulin in liquid food.
Background
Patulin is a toxic secondary metabolite of some fungal species such as Aspergillus (Aspergillus), Penicillium (Penicillium), Byssochlamys (myceliophthora) and the like. Strains that metabolically produce patulin include a.clavatus, a.giganteus, a.longivisica, p.clavigerum, p.carreum, p.coprobium, p.concentricum, p.dipodomycola, p.glandicola, p.gladioli, p.griseofulvum, p.expansum, p.marinum, p.sclerotigum, p.paneum, and p.vulpinum.
Patulin can contaminate a variety of food products, such as fruits, vegetables, grains, cheese, etc., which can pose a variety of health risks to humans and animals if passed into the body. Several toxicological studies have shown that patulin can invade the skin, kidneys, liver, gastrointestinal tract and nervous system, causing damage to tissues and organs of the human body. In some zoological studies, patulin also has acute and subacute toxicity, immunotoxicity, teratogenicity, mutagenicity, carcinogenicity, and the like.
Currently, the detection method of patulin is mainly a chromatography method, such as thin layer chromatography, high performance liquid chromatography and gas chromatography. Although the chromatography has high sensitivity and high specificity for detecting the clavulanin, the chromatography needs complicated purification steps, a large amount of raw materials and expensive instruments, is not suitable for field real-time detection in actual occasions and does not meet the requirements of the food industry.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and develop a rapid and sensitive detection method for patulin. The invention provides an patulin fluorescence detection method based on immunoliposomes, which is used for developing patulin immunoliposomes by using rabbit anti-patulin BSA antibodies and is applied to the detection of patulin.
The technical scheme of the invention is summarized as follows:
A. preparing a fluorescent dye encapsulated liposome;
B. preparing an anti-aspergillus clavatus-BSA IgG encapsulated immunoliposome;
C. detecting patulin by using a fluorescence method based on immunoliposomes;
D. specificity test of a patulin fluorescence detection method based on immunoliposomes;
and E, detecting patulin by an HPLC method.
The preparation of the fluorescent dye encapsulated liposome in the step A comprises the following steps:
and dissolving the DPPE and the SATA in a chloroform solution, and carrying out ultrasonic treatment to form DPPE-SATA. Dissolving DPPE-SATA, DPPC, DPPG and cholesterol in a mixed solution of chloroform and methanol, sonicating to form lipids, adding a sealant, sonicating. The organic solvent was removed by evaporation, leaving a deep purple gel-like suspension. The lipid suspension is added with the sealant and sonicated, followed by repeated vortexing, evaporation and sonication until the mixture becomes a homogeneous suspension. The suspension was extruded through a membrane filter to obtain liposomes of uniform size. Liposomes encapsulating SRB uniformly in size were dialyzed overnight against dialysis membrane in HEPES buffer.
The chloroform solution contained 0.7% triethylamine.
The sealant was SRB dispersed in 0.02M HEPES buffer, where the concentration of SRB was 100mM and the pH of the sealant was 7.5.
The HEPES buffer concentration of 0.01M, pH7.5, containing 0.2M NaCl and 0.01% NaN 3.
The step of preparing immunoliposome in the step B is as follows:
to prepare IgG-labeled liposomes, sulfo-KMUS was added to an anti-patulin-BSA IgG solution and the reaction was shaken to prepare derivatized IgG with maleimide groups. The derivatized anti-patulin-BSA IgG was dialyzed. Meanwhile, hydroxylamine hydrochloride was dissolved in HEPES solution and mixed with liposome solution to remove acetylthioacetic acid groups on the liposome nanoparticles. The thiol-containing liposome solution was adjusted to pH 7.0 and then mixed with derivatized IgG bearing maleimide groups. The mixture was incubated at room temperature for 4 hours after nitrogen purging, and then overnight at 4 ℃ in the dark. Ethylmaleimide was added to the reaction and gently shaken at room temperature to quench unreacted thiol groups. The mixture was then filtered and dialyzed against 0.02M TBS solution.
The concentration of sulfo-KMUS is 2mg/mL, and the sulfo-KMUS is dissolved in a mixed solvent of DMSO and methanol (2:1, v/v).
The dialysis conditions of the derivatized anti-patulin-BSA IgG are as follows: dialyzed overnight against light in HEPES solution at 0.02M, pH 7.0, containing 0.15M NaCl and 0.01% NaN 3.
The HEPES solution was 0.1M in concentration, pH7.5, and contained 25mM EDTA.
The ethyl maleimide concentration is 100mM, dissolved in 0.02M TBS solution, TBS solution pH 7.0.
The filtration conditions of the mixture are as follows: sepharose CL-4B column, equilibrated with 0.02M TBS.
In the step C, the step of detecting patulin by using a fluorescence method based on immunoliposomes comprises the following steps:
the prepared immunoliposome stock solution was diluted with TBS buffer for future use. First, anti-patulin-BSA IgG was dropped onto a 96-well microplate and incubated for 2 h. The 96-well plate was then washed with Phosphate Buffered Saline (PBST). And adding the diluted patulin solution into a washed 96-well microplate, and incubating for 1 h. The diluted immunoliposome solution was added to a 96-well microplate washed with PBST, and after incubation for 1h, the plate was washed three times with PBST. Finally, OG solution is added to a 96-well microplate to dissolve the immunoliposomes and release the fluorescent agent SRB. The fluorescence intensity signal is measured. Serial dilutions of patulin were analyzed to determine the limit of detection.
The TBS buffer solution has a concentration of 0.01M, contains 0.04M sucrose, and has a dilution ratio of 1:10 to the prepared immunoliposome stock solution.
The Phosphate Buffer (PBST) concentration is 0.01M, containing 0.05% Tween 20.
The incubation conditions were all 4 ℃.
The fluorescence detection conditions are as follows: the excitation wavelength is 550nm, and the emission wavelength is 585 nm; the instrument model was Infinite M200, Tecan, Mannedorf, Switzerland.
By adopting the technical scheme, the fluorescence intensity signal is measured under the conditions of the excitation wavelength of 550nm and the emission wavelength of 585nm, and the rapid detection of patulin can be realized.
The invention has the following beneficial effects:
compared with the existing HPLC or HPLC related methods, the fluorescence detection method based on the immunoliposome is very rapid, can detect the patulin in the apple juice sample within 3 hours, and realizes the rapid detection of the patulin. In addition, the fluorescence detection method based on the immunoliposome is simple and easy to implement, does not need complex pretreatment steps, does not need organic solvent extraction and washing processes, and is environment-friendly. The fluorescence detection method based on the immunoliposome is rapid, simple and convenient, and the detection limit is 3.15 mug/L.
Drawings
FIG. 1: a fluorescence detection standard curve of patulin, wherein the linear range is 0-150 mug/L; the standard curve equation is that Y is 4.3102X +55.432, R2＝0.9641；
FIG. 2: fluorescence detection maps of patulin, bovine serum albumin and ochratoxin A based on immunoliposomes.
Detailed Description
The present invention is described in further detail below with reference to specific examples.
Example 1: preparation of fluorescent dye-encapsulated liposomes
DPPE, DPPC, DPPG, cholesterol and SRB are used as raw materials to prepare the fluorescent dye encapsulated liposome. Briefly, DPPE (7.2. mu. mol) and SATA (14.3. mu. mol) were dissolved in 1mL of 0.7% triethylamine in chloroform and sonicated for 1 minute under a nitrogen flush to form DPPE-SATA. DPPC (40.3. mu. mol), DPPG (4.2. mu. mol) and cholesterol (40.9. mu. mol) were dissolved in a mixed solution of 3mL of chloroform and 0.5mL of methanol, sonicated at 45 ℃ under a nitrogen purge for 1min to form lipids, and then immediately 2mL of a sealant (100mM SRB in 0.02M HEPES buffer; pH7.5) was added to the lipid mixture, sonicated at 45 ℃ under a nitrogen purge for 3 min. Thereafter, the organic solvent was removed by evaporation at 45 ℃ leaving a deep purple gel-like suspension. A further 2mL of sealant was added to the lipid suspension and sonication was performed for 1min, after which vortexing, evaporation and sonication were repeated until the mixture became a homogeneous suspension. Liposomes of uniform size were obtained by extruding the suspension through 0.8 μm and 0.4 μm membrane filters in sequence. The liposomes encapsulating SRB with uniform particle size were dialyzed overnight against a dialysis membrane in 0.01M HEPES buffer containing 0.2M NaCl and 0.01% NaN3(pH 7.5).
Example 2: preparation of anti-Aspergillus clavatus-BSA IgG encapsulated immunoliposome
To prepare IgG-labeled liposomes, sulfo-KMUS (2mg/mL) dissolved in a mixed solvent of DMSO: methanol (2:1, v/v) was added to 1mL of anti-patulin-BSA IgG solution and reacted at room temperature at 70rpm for 3h to prepare derivatized IgG carrying a maleimide group. The derivatized anti-patulin-BSA IgG was dialyzed overnight at 4 ℃ against light in a 0.02M HEPES solution containing 0.15M NaCl and 0.01% NaN3(pH 7.0) using a dialysis membrane. Meanwhile, 30. mu. L0.5M hydroxylamine hydrochloride was dissolved in 0.1M HEPES solution containing 25mM EDTA (pH7.5) and mixed with 300. mu.L of the liposome solution to remove the acetylthioacetic acid group on the liposome nanoparticles. After purging with nitrogen for 1min, the liposome solution was reacted at room temperature for 2 h. The thiol-containing liposome solution was adjusted to pH 7.0 with 0.5M HEPES solution and mixed with derivatized IgG bearing maleimide groups. The mixture was purged with nitrogen for 1min and incubated at room temperature for 4h, then overnight at 4 ℃ in the absence of light. Ethylmaleimide dissolved in 0.02M TBS (pH 7.0) at a concentration of 100mM was added to the reaction and unreacted thiol groups were quenched by gentle shaking (70rpm) at room temperature for 30 min. The mixture was then filtered on a Sepharose CL-4B column equilibrated in 0.02M TBS. The collected immunoliposome solution was dialyzed against a dialysis membrane in 0.02M TBS solution.
The characteristics of the liposomes and anti-A.clavuligerus-BSA IgG encapsulated immunoliposomes are shown in Table 1.
TABLE 1 characterization of liposomes and immunoliposomes
Figure BDA0003359024850000061
Figure BDA0003359024850000071
All experiments were repeated three times and the data represent the mean standard deviation.
As can be seen from the data in Table 1, the liposome particle size increased, indicating that immunoliposomes were successfully prepared by coating liposomes with anti-Aspergillus clavus-BSA IgG. Assuming an initial concentration of 100mM SRB, the internal volumes of both the liposomes and immunoliposomes developed were 3.03X 10-12. mu.L, with SRB encapsulated at a concentration of 3.03X 10-13. mu. mol. As shown in Table 1, the polydispersity indexes of the prepared liposome and immunoliposome are 0.19 + -0.00 and 0.17 + -0.01, respectively, and the low polydispersity index indicates that the prepared liposome and immunoliposome have good stability. The zeta potential of the nanoparticles reflects the potential stability of the colloidal system. When the particles in the suspension have a large negative or positive zeta potential, the particles will repel each other, stabilizing the suspension. As shown in table 1, both liposomes and immunoliposomes have a negative zeta potential, indicating that the particles in suspension have resistance to clumping together. The experimental result shows that the prepared liposome and the immunoliposome are stable and uniform.
Example 3: fluorescence method for detecting patulin based on immunoliposome
mu.L of anti-patulin-BSA IgG (1. mu.g/mL) was added dropwise to a 96-well microplate and incubated at 4 ℃ for 2 h. The 96-well plates were washed with 200. mu.L of 0.01M Phosphate Buffered Saline (PBST) containing 0.05% Tween 20. mu.L of the diluted patulin solution was added to the washed 96-well microplate and incubated at 4 ℃ for 1 h. mu.L of the diluted immunoliposome solution was added to a 96-well microplate washed with 200. mu.L of PBST. The immunoliposome was cultured in a 96-well microplate at 4 ℃ for 1 hour, and then the 96-well microplate was washed three times with 200. mu.L of PBST. Finally, 200 μ L of OG solution was added to a 96-well microplate to dissolve the immunoliposomes releasing the fluorescent agent SRB. The fluorescence intensity signal was measured at an excitation wavelength of 550nm and an emission wavelength of 585 nm.
As a result, as shown in FIG. 1, when the concentration of patulin was increased from 0. mu.g/L to 150. mu.g/L, the fluorescence intensity was sharply increased. The fluorescence intensity signal has good linear relation with the patulin concentration, R20.9641, this shows that the inventive immunoliposome-based patulin fluorescence detection method can give quantitative analysis results. However, when the concentration of patulin was higher than 150. mu.g/L, the fluorescence intensity did not increase any more, indicating that the established method can detect but does not give a quantitative analysis result for detecting patulin because there is no linearity when the concentration of patulin is higher than 150. mu.g/L.
Example 4: specificity of immunoliposome-based fluorescence detection
A solution of ochratoxin A dissolved in 0.01M PBS was diluted to concentrations of 10. mu.g/L, 50. mu.g/L and 100. mu.g/L. These various concentrations of ochratoxin a are measured by the inventive immunoliposome-based fluorescence detection method. Also, to confirm the cross-reactivity of BSA to the inventive detection method, different concentrations of BSA in apple juice were also determined by the inventive immunoliposome-based fluorescence detection method.
The specificity of the immunoliposome-based patulin fluorescence detection method was examined using ochratoxin a as a control toxin. As shown in FIG. 2, when the concentration of ochratoxin A was increased from 0. mu.g/L to 200. mu.g/L, there was no change in the fluorescence signal, indicating that the method did not cross-react with ochratoxin A. On the other hand, BSA was part of the immunogen and cross-reactivity of BSA with the inventive immunoliposome-based fluorescence detection method was examined. As shown in FIG. 2, the immunoliposome-based fluorescence assay of the present invention has cross-reactivity with BSA, which may limit the application of the developed method for detecting patulin when BSA is present in a food sample.
Example 5: determination of patulin by HPLC method
Apple juice samples artificially contaminated with 10. mu.g/L, 50. mu.g/L, 100. mu.g/L, 200. mu.g/L and 500. mu.g/L patulin were analyzed by HPLC. First, patulin was extracted from an artificially contaminated apple juice sample with ethyl acetate, and the ethyl acetate fraction was treated with 0.5% sodium carbonate and passed through anhydrous sodium sulfate using a buchner funnel. The filtrate was dried under vacuum and dissolved in acetic acid solution (pH 4.0) and then filtered with a 0.22 μm syringe filter for HPLC analysis in an Ultimate 3000(Thermo Scientific; Waltham, MA, USA) equipped with a UV detector. mu.L of the sample was injected onto a C18 column (5 μm pore size, 4.6mm inner diameter × 250mm length) (Waters; Milford, Mass., USA) and eluted using 5% acetonitrile (v/v) at a constant flow rate of 0.5 mL/min. Patulin is detected at 276nm and quantified by comparing the signal from the sample to that from a known concentration of patulin.
Table 2 comparison of the recovery of patulin from artificially contaminated apple juice by immunoliposome-based fluorescence detection and HPLC detection.
Figure BDA0003359024850000091
All experiments were repeated three times and the data represent the mean standard deviation.
As shown in Table 2, when the spiked concentrations of patulin were 10. mu.g/L, 50. mu.g/L, 100. mu.g/L, 200. mu.g/L and 500. mu.g/L, the concentrations of patulin were 6.19. + -. 0.31. mu.g/L, 35.63. + -. 0.62. mu.g/L, 74.38. + -. 6.88. mu.g/L, 151.88. + -. 3.12. mu.g/L and 481.88. + -. 1.31. mu.g/L, respectively, and the recoveries were 61.88. + -. 0.03%, 71.25. + -. 0.01%, 74.38. + -. 0.07%, 75.02% and 96.38. + -. 0.03%, respectively. In HPLC analysis of patulin, organic solvents during extraction will significantly affect recovery. Low recovery indicates loss of patulin during extraction. When the standard addition concentrations of patulin are 10 mug/L, 50 mug/L and 100 mug/L, the recovery rates of the immunoliposome-based fluorescence detection method are 73.24 +/-0.06%, 101.73 +/-1.55% and 98.74 +/-4.88%, respectively, which indicates that the method is sensitive and reliable for detecting patulin. When the spiked concentrations of patulin were 200. mu.g/L and 500. mu.g/L, the recovery rates were 69.97. + -. 0.17% and 32.7. + -. 1.31%, which is consistent with the method for detecting patulin in a pure solution, which has a good linear relationship in the range of patulin concentrations of 0. mu.g/L to 150. mu.g/L. However, the linearity of the process is reduced in both pure solutions and artificially contaminated apple juice solutions when the patulin concentration is higher than 200. mu.g/L.

Claims (10)

1. The immunoliposome coated with the fluorescent dye is characterized in that the immunoliposome is prepared by the following steps:
1) dissolving DPPE and SATA in a chloroform solution, and carrying out ultrasonic treatment to form DPPE-SATA; dissolving DPPE-SATA, DPPC, DPPG and cholesterol in a mixed solution of chloroform and methanol, and carrying out ultrasonic treatment to form liposome; the liposome is encapsulated by the fluorescent dye to obtain the fluorescent dye encapsulated liposome;
2) modifying the liposome encapsulated by the fluorescent dye by using the antibody derived from the sulfo-KMUS to obtain the immune liposome encapsulating the fluorescent dye.
2. The fluorescent dye-encapsulated immunoliposome of claim 1, wherein the chloroform solution in step 1) contains 0.7% triethylamine.
3. The immunoliposome encapsulating a fluorescent dye according to claim 1, wherein the fluorescent dye of step 1) is dispersed in 0.02M HEPES buffer at a concentration of 100mM and pH 7.5.
4. The fluorescent dye-encapsulated immunoliposome of claim 3, wherein said HEPES buffer has a concentration of 0.01M, a pH of 7.5, and comprises 0.2M NaCl and 0.01% NaN3。
5. The immunoliposome encapsulated with a fluorescent dye of claim 1, wherein the specific steps of step 2) are:
1) adding sulfo-KMUS into an anti-patulin-BSA IgG solution, carrying out a shaking reaction to prepare a derivatized IgG with a maleimide group, and dialyzing the derivatized anti-patulin-BSA IgG;
2) dissolving hydroxylamine hydrochloride in HEPES solution, and mixing with liposome solution to remove acetylthioacetic acid groups on the liposome nanoparticles;
3) adjusting the pH of the liposome solution containing sulfydryl to 7.0, and mixing with the derivatized IgG with maleimide groups;
4) the mixture is cultured for 4 hours at room temperature after being blown by nitrogen, and then cultured overnight at 4 ℃ in a dark condition;
5) adding ethylmaleimide to the reaction in step 4), gently shaking at room temperature to quench unreacted thiol, and then, filtering the mixture and dialyzing against 0.02M TBS solution to prepare immunoliposome encapsulating a fluorescent dye.
6. The fluorescent dye-encapsulated immunoliposome according to claim 5, wherein the concentration of sulfo-KMUS in step 1) is 2mg/mL, and is dissolved in a mixed solvent of DMSO and methanol (2:1, v/v), wherein DMSO: the volume ratio of the methanol is 2: 1; the conditions for dialysis of derivatized anti-patulin-BSA IgG were: dialyzing overnight in HEPES solution with light shielding, wherein the HEPES solution has a concentration of 0.02M and a pH of 7.0, and contains 0.15M NaCl and 0.01% NaN3。
7. The fluorescent dye-encapsulated immunoliposome of claim 5, wherein the HEPES solution in step 2) has a concentration of 0.1M, a pH of 7.5, and contains 25mM EDTA; in the step 3), the concentration of the ethylmaleimide is 100mM, and the ethylmaleimide is dissolved in a TBS solution of 0.02M, wherein the pH of the TBS solution is 7.0; step 5) the mixture was filtered on a Sepharose CL-4B column and equilibrated with 0.02M TBS.
8. Use of the fluorescent dye-encapsulated immunoliposome of claims 1-7 in the fluorescence detection of patulin.
9. The application of claim 8, wherein the application comprises the steps of:
1) diluting the prepared immunoliposome stock solution with TBS buffer solution for later use;
2) dripping anti-patulin-BSA IgG onto a 96-well microplate, incubating for 2 hours, and washing the 96-well microplate by using phosphate buffer solution PBST;
3) adding the diluted patulin solution into a washed 96-hole microporous plate, incubating for 1h, adding the diluted immunoliposome solution into the 96-hole microporous plate washed by PBST, incubating for 1h, and washing the microtiter plate for three times by PBST;
4) and adding the OG solution into a 96-well micro-porous plate to dissolve the immunoliposome to release the fluorescent agent SRB, and measuring a fluorescence intensity signal.
10. The use according to claim 9, wherein in step 1) the TBS buffer solution is 0.01M, contains 0.04M sucrose, and is diluted to the prepared immunoliposome stock solution in a ratio of 1: 10; in the step 2), the concentration of the phosphate buffer PBST is 0.01M, and the phosphate buffer PBST contains 0.05 percent of Tween 20; the incubation conditions are all 4 ℃, and the fluorescence detection conditions are as follows: the excitation wavelength was 550nm and the emission wavelength was 585 nm.
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