CN104458615A - Preparation method of photonic crystal total-reflection layer and total-reflection layer-based fast detector for total number of bacteria - Google Patents
Preparation method of photonic crystal total-reflection layer and total-reflection layer-based fast detector for total number of bacteria Download PDFInfo
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- CN104458615A CN104458615A CN201410728254.5A CN201410728254A CN104458615A CN 104458615 A CN104458615 A CN 104458615A CN 201410728254 A CN201410728254 A CN 201410728254A CN 104458615 A CN104458615 A CN 104458615A
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Abstract
The invention provides a preparation method of a photonic crystal total-reflection layer and a total-reflection layer-based fast detector for the total number of bacteria, relates to the bacteria number detection technology and aims at solving the problems that when a conventional rapid bacteria detection method is adopted, the large size and the high price can be caused due to the adoption of an optical grating for generating monochromatic light with the wavelength of 600nm. A photonic crystal microsphere is adopted to prepare an ethanol-water suspension, glucose is added in the ethanol-water suspension to obtain a mixture, the mixture is cultured on a glass plate at 70 DEG C, and the glass plate is taken out and encapsulated after 4 hours, and therefore, the photonic crystal total-reflection layer is obtained; the photonic crystal total-reflection layer is adopted to replace the optical grating to obtain the monochromatic light with the wavelength of 600nm, the monochromatic light is applied to the rapid bacteria number detection, the instrument stability is good, the size is small, the detector can be carried conveniently, and the cost is low. The fast detector is suitable for the bacteria number detection.
Description
Technical field
The present invention relates to bacterial number detection technique.
Background technology
In existing bacterium fast speed detection method, one is ATP (atriphos) biloluminescence method, the main shortcoming of the method to distinguish microorganism and non-microorganism ATP, can not distinguish the kind of microorganism specifically, and some ion that sample itself and ATP extraction agent etc. contain can cause interference to the mensuration of ATP and suppress luminous function.Meanwhile, the method needs suitable ATP extraction agent, needs the operation of professional, and by free ATP with somaticly affect comparatively large, enzymatic reaction influence factor is many, causes testing result precision not high enough, and expensive reagents, add testing cost.Another kind of bacterium fast speed detection method is, adopt grating to obtain the monochromatic light that wavelength is 600nm, 600nm monochromatic light enters testing sample, after testing sample outgoing, utilize photodetector to detect, after result of detection is processed, obtain the total number of bacteria in testing sample.This method does not need chemical reagent, does not relate to enzymatic reaction, and the accuracy of thus measuring is higher, and the stability of whole device is also higher, but grating is bulky, is inconvenient to carry, and gratings expensive, add the cost of whole detecting instrument.
Summary of the invention
The object of the invention is to solve conventional bacterium fast speed detection method owing to adopting grating to produce the monochromatic light that wavelength is 600nm, and grating is not only bulky causes carrying inconvenience, and it is expensive, add the problem of the cost of detecting instrument, provide a kind of photonic crystal total reflection layer preparation method and based on this totally reflected total number of bacteria fast detector.
Photonic crystal total reflection layer preparation method of the present invention comprises the following steps:
The photon crystal micro-ball that step one, preparation monodispersity are homogeneous, described photonic crystal can produce photon band gap at 600nm place;
Step 2, by described photon crystal micro-ball through centrifugal, washing and drying after, be configured to the ethanol aqueous suspension that photon crystal micro-ball concentration is 3% to 6%, in this ethanol aqueous suspension, the ratio shared by the volume of ethanol is 15% to 25%;
Step 3, in described ethanol aqueous suspension, add glucose, and stir, the mass ratio of the glucose added and ethanol aqueous suspension is 0.5:100 to 2.5:100;
Step 4, be incubated on glass plate by the ethanol aqueous suspension after stirring under constant temperature 65 DEG C to 75 DEG C conditions, incubation time is 3.5 to 4.25 hours, cultivates after terminating and glass plate is taken out encapsulation, obtain photonic crystal total reflection layer.
Based on the totally reflected total number of bacteria fast detector of above-mentioned photonic crystal, this total number of bacteria fast detector comprises light source, photonic crystal total reflection layer, absorption cell, photomultiplier and reading device, the light that light source sends is radiated on photonic crystal total reflection layer, absorption cell is entered after the reflection of photonic crystal total reflection layer, be radiated at the photosurface of photomultiplier from the light of absorption cell outgoing, photomultiplier converts light signal to electric signal, and this electric signal is sent to reading device.
Photonic crystal total reflection layer of the present invention can be totally reflected the light that wavelength is 600nm, to the wavelength transmission beyond 600nm, can be used for producing the monochromatic light of 600nm.During based on this photonic crystal totally reflected total number of bacteria fast detector bacterial detection sum, photonic crystal total reflection layer is totally reflected the light that the wavelength that light source sends is 600nm, light after reflection enters the absorption cell that testing sample is housed, the quantity of bacterium testing sample can be reflected from the light of absorption cell outgoing, therefore, after utilizing photomultiplier to convert light signal to electric signal, the sum of bacterium in testing sample can directly be read by reading device.Cleaning Principle is different from conventional ATP biloluminescence method, and testing process does not relate to enzymatic reaction, and the good stability of detector self, in testing process, disturbing factor is few, and accuracy of detection is high; Do not need the chemical reagent of various costliness, reduce cost; Simple to operate, detection time is short.Reach 0.9 with the dressing plate bacterium colony method degree of correlation, (ATP luminescence method and the dressing plate bacterium colony method degree of correlation are 0.85), be always consuming timely no more than 30min.Compared with grating, photonic crystal total reflection layer is adopted to obtain 600nm monochromatic light, not only compact structure, easy to carry, and also photonic crystal total reflection layer preparation method is simple, reduces instrument cost.
Accompanying drawing explanation
Fig. 1 is the principle schematic based on the totally reflected total number of bacteria fast detector of photonic crystal of the present invention.
Embodiment
Embodiment one: the photonic crystal total reflection layer preparation method described in present embodiment comprises the following steps:
The photon crystal micro-ball that step one, preparation monodispersity are homogeneous, described photonic crystal can produce photon band gap at 600nm place;
Step 2, by described photon crystal micro-ball through centrifugal, washing and drying after, be configured to the ethanol aqueous suspension that photon crystal micro-ball concentration is 3% to 6%, in this ethanol aqueous suspension, the ratio shared by the volume of ethanol is 15% to 25%;
Step 3, in described ethanol aqueous suspension, add glucose, and stir, the mass ratio of the glucose added and ethanol aqueous suspension is 0.5:100 to 2.5:100;
Step 4, be incubated on glass plate by the ethanol aqueous suspension after stirring under constant temperature 65 DEG C to 75 DEG C conditions, incubation time is 3.5 to 4.25 hours, cultivates after terminating and glass plate is taken out encapsulation, obtain photonic crystal total reflection layer.
Described encapsulation refers to the upper surface covering thin film at glass plate, and glass plate sealing be hedged off from the outer world, this film is thoroughly high to the light of 600nm.
Present embodiment adopts the photonic crystal that can produce photon band gap at 600nm place to prepare photonic crystal total reflection layer, this total reflection layer can be totally reflected the light that wavelength is 600nm, this photonic crystal total reflection layer is used for total number of bacteria to detect, enzymatic is not needed to reflect, accuracy of detection can be improved, and compared with grating, cost reduces greatly, the quick checkout equipment of total number of bacteria that this photonic crystal total reflection layer is highly suitable for miniaturization low cost.
Embodiment two: present embodiment is the further restriction to the photonic crystal total reflection layer preparation method described in embodiment one, in present embodiment, the photonic crystal described in step one is opal structural or photonic crystal with inverse opal structure.
With the SiO of opal structural
2photonic crystal is example, and microsphere diameter is 288nm.The photonic crystal of different structure, its diameter has specific relation with the wavelength producing photon band gap, searches by reference book.
Embodiment three: present embodiment is the further restriction to the photonic crystal total reflection layer preparation method described in embodiment one, in the ethanol aqueous suspension described in step 2, photon crystal micro-ball concentration is 4%.
Embodiment four: present embodiment is the further restriction to the photonic crystal total reflection layer preparation method described in embodiment one, in the ethanol aqueous suspension described in step 2, the ratio shared by the volume of ethanol is 20%.
Embodiment five: present embodiment is the further restriction to the photonic crystal total reflection layer preparation method described in embodiment one, in step 3, the mass ratio of glucose and ethanol aqueous suspension is 2:100.
Embodiment six: present embodiment is the further restriction to the photonic crystal total reflection layer preparation method described in embodiment one, in step 4, constant temperature is 70 DEG C.
Embodiment seven: present embodiment is the further restriction to the photonic crystal total reflection layer preparation method described in embodiment six, in step 4, incubation time is 4 hours.
Embodiment eight: present embodiment is the further restriction to the photonic crystal total reflection layer preparation method described in embodiment one, in step one, utilizes stober legal system for the homogeneous photon crystal micro-ball of monodispersity.
Embodiment nine: composition graphs 1 illustrates present embodiment, described in present embodiment based on the totally reflected total number of bacteria fast detector of photonic crystal, this detector comprises light source 1, photonic crystal total reflection layer 2, absorption cell 3, photomultiplier 4 and reading device 5, the light that light source 1 sends is radiated on photonic crystal total reflection layer 2, absorption cell 3 is entered after photonic crystal total reflection layer 2 reflects, be radiated at the photosurface of photomultiplier 4 from the light of absorption cell 3 outgoing, photomultiplier 4 converts light signal to electric signal, and this electric signal is sent to reading device 5.
Grow in microorganism liquid medium within, due to the increase of protoplasm content, cause increasing of culture turbidity, light absorption value becomes positive correlation within the specific limits with cell density, can Accurate Measurement thalline quantity under 600nm condition.Therefore in present embodiment, the wavelength of the light that light source 1 sends need contain 600nm, adopts tungsten lamp.Photonic crystal total reflection layer 2 is used for reflecting the light that wavelength is 600nm, places detected sample in absorption cell 3.The light of the 600nm after photonic crystal total reflection layer 2 reflects is after absorption cell 3, and detect with photomultiplier 4, detectable signal is sent to reading device 5, can directly read total number of bacteria in absorption cell 3 from reading device 5.Before detection, need detector zeroing, adjusting zero method is identical with above-mentioned testing process, just the detected sample in absorption cell 3 is replaced with sterile sampling, described sterile sampling be detected sample after treatment by removal of bacteria, but all the other compositions remain unchanged.
The total number of bacteria fast detector described in present embodiment is adopted to detect total number of bacteria in fermentation liquor.After start, tungsten light source preheating 15min, the light that light source is launched is stablized, photonic crystal total reflection layer 2 produces stable total reflection monochromatic light, blank zeroing is carried out with the fermentation liquor not connecing bacterium, in glass cuvette, add the brevibacterium flavum glutami acid fermentation liquor in 3.5mL different fermentations period, put in absorption cell 3, directly can obtain the total number of bacteria value of this fermentation liquor from reading device.
Adopt the total number of bacteria in above-mentioned total number of bacteria fast detector detection water body.Instrument is started shooting, tungsten light source preheating 15min, the light that light source is launched is stablized, and photonic crystal total reflection layer 2 produces stable total reflection monochromatic light, is carried out by water sample to be measured filtering, centrifugal purification process, obtain the water body solution clarified, carry out blank zeroing with this, the water body solution getting another part of clarification cultivates 20min under 37 DEG C of conditions, gets 3.5mL and adds in glass cuvette and detect, put in absorption cell, directly can obtain water body total number of bacteria value from reading device.
Adopt the total number of bacteria in above-mentioned total number of bacteria fast detector detection probiotic beverage.Instrument is started shooting, tungsten light source preheating 15min, the light that light source is launched is stablized, photonic crystal total reflection layer 2 produces stable total reflection monochromatic light, by capable for probiotic beverage to be measured filtration, centrifugal purification process, obtain the solution clarified, use normal saline dilution 1000 times again, blank zeroing is carried out with this, get another part and cultivate 20min under 37 DEG C of conditions, get 3.5mL to add in glass cuvette and detect, put in absorption cell, directly can obtain probiotic beverage total number of bacteria value from reading device.
Total number of bacteria fast detector described in present embodiment adopts photonic crystal total reflection layer 2 to substitute grating to obtain 600nm monochromatic light, not only compact, easy to carry, and cost also reduces greatly.When carrying out total number of bacteria detection, do not relate to enzymatic reaction, and the good stability of detector self, in testing process, disturbing factor is few, and accuracy of detection is high; Do not need the chemical reagent of various costliness, reduce cost; Simple to operate, detection time is short.Reach 0.9 with the dressing plate bacterium colony method degree of correlation, be always consuming timely no more than 30min.
The stability of the total number of bacteria fast detector described in present embodiment is detected, get 0.5% p styrene sulfonic acid to receive solution 3.5ml and add in cuvette, cuvette is put into the device absorption cell 3 of detector, observe sample light absorption value change in 3h, result shows that in 180 minutes, light absorption value change is 0.02 to the maximum, and the impact of stability of instrument on testing result is less than 1%.Stability of instrument is good.
Embodiment ten: present embodiment is to the further restriction based on photonic crystal totally reflected total number of bacteria fast detector described in embodiment nine, in present embodiment, described photonic crystal total reflection layer 2 is the SiO of opal structural
2photonic crystal total reflection layer.
SiO
2photonic crystal stable performance, price is low, is applicable to prepare photonic crystal total reflection layer.
Claims (10)
1. photonic crystal total reflection layer preparation method, is characterized in that, the method comprises the following steps:
The photon crystal micro-ball that step one, preparation monodispersity are homogeneous, described photonic crystal can produce photon band gap at 600nm place;
Step 2, by described photon crystal micro-ball through centrifugal, washing and drying after, be configured to the ethanol aqueous suspension that photon crystal micro-ball concentration is 3% to 6%, in this ethanol aqueous suspension, the ratio shared by the volume of ethanol is 15% to 25%;
Step 3, in described ethanol aqueous suspension, add glucose, and stir, the mass ratio of the glucose added and ethanol aqueous suspension is 0.5:100 to 2.5:100;
Step 4, be incubated on glass plate by the ethanol aqueous suspension after stirring under constant temperature 65 DEG C to 75 DEG C conditions, incubation time is 3.5 to 4.25 hours, cultivates after terminating and glass plate is taken out encapsulation, obtain photonic crystal total reflection layer.
2. photonic crystal total reflection layer preparation method according to claim 1, it is characterized in that, the photonic crystal described in step one is opal structural or photonic crystal with inverse opal structure.
3. photonic crystal total reflection layer preparation method according to claim 1, is characterized in that, in the ethanol aqueous suspension described in step 2, photon crystal micro-ball concentration is 4%.
4. photonic crystal total reflection layer preparation method according to claim 1, is characterized in that, in the ethanol aqueous suspension described in step 2, the ratio shared by the volume of ethanol is 20%.
5. photonic crystal total reflection layer preparation method according to claim 1, is characterized in that, in step 3, the mass ratio of glucose and ethanol aqueous suspension is 2:100.
6. photonic crystal total reflection layer preparation method according to claim 1, is characterized in that, in step 4, constant temperature is 70 DEG C.
7. photonic crystal total reflection layer preparation method according to claim 6, is characterized in that, in step 4, incubation time is 4 hours.
8. photonic crystal total reflection layer preparation method according to claim 1, is characterized in that, in step one, utilizes stober legal system for the homogeneous photon crystal micro-ball of monodispersity.
9. based on the totally reflected total number of bacteria fast detector of photonic crystal, comprise light source (1), absorption cell (3), photomultiplier (4) and reading device (5), it is characterized in that, it also comprises photonic crystal total reflection layer (2), the light that light source (1) sends is radiated on photonic crystal total reflection layer (2), absorption cell (3) is entered after photonic crystal total reflection layer (2) reflection, be radiated at the photosurface of photomultiplier (4) from the light of absorption cell (3) outgoing, photomultiplier (4) converts light signal to electric signal, and this electric signal is sent to reading device (5).
10. according to claim 9ly to it is characterized in that based on the totally reflected total number of bacteria fast detector of photonic crystal, the SiO that described photonic crystal total reflection layer (2) is opal structural
2photonic crystal total reflection layer.
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