CN106140450A - Microsphere separation device and separation method thereof - Google Patents

Abstract

The invention provides a microsphere separation device and separation method with high separation precision and high yield, which are suitable for large-scale application, comprising a barrel body and a base arranged under the barrel body, and the side wall of the barrel body is provided with The microsphere exit hole, the sampling microsphere exit hole and the control marking line are marked, and the microsphere exit hole and the sampling microsphere exit hole are respectively provided with a first branch pipe and a second branch pipe protruding outward relative to the barrel. Utilize the sedimentation gradient method combined with the device of the present invention to separate and purify polymer microspheres with highly uniform particle size, which can be applied to the separation of microspheres of any scale; and the separation accuracy is high and the yield is high; there are microsphere exit holes and Sampling inspection of the microsphere exit holes facilitates the tracking of the separation process and the collection at the end of the separation.

Description

A microsphere separation device and separation method thereof

technical field

The invention relates to a separation device, in particular to a microsphere separation device and a method thereof.

Background technique

Monodisperse polymer microspheres have been widely used in many fields such as bioanalysis, high-throughput detection, immunoassay and micro-total analysis system. The particle size uniformity of polymer microspheres has a great influence on its performance. Most researchers can obtain microspheres with relatively uniform particle size distribution by improving the method and controlling the process parameters. The commonly used method for preparing monodisperse microspheres is dispersion polymerization. , seed polymerization and emulsion polymerization, etc., but in fact, during the preparation of polymer microspheres, since the polymerization reaction is always carried out on the phase interface, although one mechanism is the main mechanism in the system, there are many side polymerization methods at the same time. Moreover, temperature changes and monomer absorption processes are always inevitable during the polymerization process, which will lead to a broadening of the particle size distribution of the final synthesized microspheres.

Especially, for seed polymerization, the synthesized microspheres always contain some microspheres with smaller particle size (subnuclear grade) and some microspheres with larger particle size, which seriously affects the dispersion of microspheres and limits the The application of balls, therefore, requires multiple separations to remove unwanted particle size components in the system. The commonly used centrifugal separation method is based on the different settling time of microspheres with different particle sizes in the centrifugal force field. Since it is difficult to select the verified centrifugal speed and time, it can only initially remove the oversized and very small particles in the system, and it is difficult to obtain particles. polymer microspheres with highly uniform diameter; continuous centrifugation can continuously separate and purify polymer microspheres, but its separation accuracy is not high. The yield is very low, and because the scale of the microspheres required by this method is very large, it is difficult to realize small-scale application; YoonD H et al. used the rotating flow channel separation method in the chip to realize the separation and purification of the microspheres, but it was limited to the separation scale and the size of the chip. cost, not suitable for large-scale applications. And in all the prior art, the separation process of the microspheres cannot be checked and sampled, which is not conducive to the tracking of the separation process.

In view of the above-mentioned defects, the designer is actively researching and innovating in order to create a microsphere separation device to make it more industrially useful.

Contents of the invention

In order to solve the above technical problems, the object of the present invention is to provide a microsphere separation device with high separation precision and high yield, which is suitable for large-scale application.

The microsphere separation device of the present invention includes a barrel body, the barrel body has a barrel bottom, a barrel body side wall and a barrel mouth, and the barrel body side walls are respectively provided with microsphere outlet holes and sampling microsphere outlet holes. Sampling microsphere exit holes are set on the upper side of the microsphere exit holes and both are provided with valves, the capacity of the barrel body is 1mL-10L, and the side wall of the barrel body is also provided with control marking lines. The height h3 _of the control marking line can be obtained by the following formula:

$\mathrm{<span\; class="notranslate">\; h</span>}\mathrm{<span\; class="notranslate">\; 3</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; k</span>}<span\; class="notranslate">\; \&CenterDot;</span>\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; \&CenterDot;</span>\frac{{\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; D.</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; \&rho;</span>}}_{\mathrm{<span\; class="notranslate">\; m</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; \&rho;</span>}}_{\mathrm{<span\; class="notranslate">\; D.</span>}}<span\; class="notranslate">\; )</span>}{{\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; L</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; \&rho;</span>}}_{\mathrm{<span\; class="notranslate">\; m</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; \&rho;</span>}}_{\mathrm{<span\; class="notranslate">\; L</span>}}<span\; class="notranslate">\; )</span>}$

Among them, x is the height of the upper limit of loading, R _D and _RL are the radii of the target microsphere and the radius of the larger microsphere respectively, ρ _m , ρ _D and ρ _L are the diameters of the sedimentation liquid, the target microsphere and the larger microsphere, respectively Apparent density, k is the separation coefficient, the value is between 0.5-1, and the selection of the k value depends on the separation requirements: if it is to obtain highly uniform microspheres, the k value can be set to 0.5; if it is to improve the separation efficiency, The k value can be set to 1.

Further, the microsphere exit hole and the sampling microsphere exit hole are respectively provided with a first branch pipe and a second branch pipe protruding outward relative to the side wall of the barrel, and the valve is arranged on the first branch pipe and the second branch pipe. Inside the two tubes.

Further, the mouth of the bucket is provided with a matching dust cover.

Furthermore, a sealing mechanism is provided at the contact between the dust-proof cover and the mouth of the barrel.

Further, the sealing mechanism is frosted glass arranged inside the dust-proof cover and outside the mouth of the barrel.

Further, the ratio of the barrel diameter d to the barrel height h is d:h=1:0.5-50, preferably d:h=2.

Further, the ratio of the distance h ₁ from the exit hole of the microsphere to the bottom of the barrel and the distance h ₂ from the exit hole of the sampling microsphere to the bottom of the barrel is h ₁ : h ₂ =1:1.5-2, preferably h ₁ :h ₂ =1:1.5, and h ₂ is between 1/10-1/3 of the barrel height h, preferably 1/4-1/3.

Further, the side wall of the barrel body is provided with a loading upper limit marking line, a loading lower limit marking line and a control marking line, and the loading capacity in the barrel is between the loading upper limit marking line and the loading lower limit marking line, preferably 60-80%. Rated volume.

Further, the height h ₃ of the control marking line is higher than h ₂ , and there can be several control line stickers marked on the barrel body that can be replaced. 1-20 control lines are marked on the sticker at the same time, and each control line is located at different heights.

The method for separating microspheres using the microsphere separation device of the present invention comprises the following steps:

(1) After mixing the microspheres and the sedimentation liquid, place them in the barrel and let them stand;

(2) When the sedimentation interface of the target microsphere is lower than the microsphere exit hole, open the valve of the microsphere exit hole, discharge the microsphere with smaller particle size, add the sedimentation liquid, repeat 3-20 times, when the target microsphere When the sedimentation interface reaches the control mark line, follow-up detection is carried out from the sampling microsphere outlet hole until the proportion of small particle size microspheres in the discharged microspheres is less than 1%;

(3) Add sedimentation liquid. When the sedimentation interface of the target microsphere reaches the control mark line, follow up and detect from sampling microsphere exit hole 4, and discharge the target microsphere from the microsphere exit hole at the same time, add sedimentation liquid, repeat 3-10 times, the proportion of smaller and larger microspheres in the microspheres separated in the last random inspection is not higher than 1%.

Firstly, the movement of microspheres in the liquid state was studied, and the formula for the movement speed of the microspheres in a constant gravity field was derived when the microspheres were suspended in the liquid phase. Refer to Zhang Qi’s doctoral thesis of Tianjin University in 2010, "Monodisperse Functional Polymer Microspheres" "Research", in which the fourth chapter clearly stated that it was found that the velocity of the microsphere is related to its own density, diameter, acceleration and the properties of the solution; on the basis of accurately measuring the density and viscosity of glycerol aqueous solution, a A method for determining the density of polymer microspheres at the milligram level; then the influence of solution properties on the signal of microspheres in flow cytometry was studied. On the basis of the liquid suspension motion of microspheres, the sedimentation gradient method was developed.

By means of the above solution, the present invention has at least the following advantages:

The sedimentation gradient method is combined with the device of the present invention to separate and purify polymer microspheres with highly uniform particle diameters. The device of the invention combined with the sedimentation gradient method can be used for the separation and purification of milligram-level quantum dot-coded fluorescent microspheres, and the analysis accuracy of the fluorescent microspheres can be improved.

The microsphere separation device of the present invention is applicable to the separation of microspheres of any scale; and has high separation precision and high yield; it is equipped with microsphere exit holes, random inspection microsphere exit holes and control lines, which is convenient for tracking and separation of the separation process End of collection.

The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and implement them according to the contents of the description, the preferred embodiments of the present invention and accompanying drawings are described in detail below.

Description of drawings

Fig. 1 is a schematic structural view of an embodiment of the device of the present invention.

detailed description

The specific implementation manners of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Referring to Fig. 1, a kind of microsphere separating device of the present invention comprises barrel body, and described barrel body has barrel bottom 2, barrel body side wall 1 and barrel mouth 13, and described barrel body side wall 1 is provided with microsphere outlet respectively. The hole 3 and the sampling microsphere outlet 4, the sampling microsphere outlet 4 is arranged on the upper side of the microsphere outlet 3 and both are provided with a valve 7, the capacity of the barrel is 1mL-10L, The staving side wall 1 is also provided with a control marking line 12, and the height h3 of the control marking line ₁₂ can be obtained by the following formula:

$\mathrm{<span\; class="notranslate">\; h</span>}\mathrm{<span\; class="notranslate">\; 3</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; k</span>}<span\; class="notranslate">\; \&Center\; Dot;</span>\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; \&CenterDot;</span>\frac{{\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; D.</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; \&rho;</span>}}_{\mathrm{<span\; class="notranslate">\; m</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; \&rho;</span>}}_{\mathrm{<span\; class="notranslate">\; D.</span>}}<span\; class="notranslate">\; )</span>}{{\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; L</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; \&rho;</span>}}_{\mathrm{<span\; class="notranslate">\; m</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; \&rho;</span>}}_{\mathrm{<span\; class="notranslate">\; L</span>}}<span\; class="notranslate">\; )</span>}$

Among them, x is the height of the upper limit of loading, R _D and _RL are the radii of the target microsphere and the radius of the larger microsphere respectively, ρ _m , ρ _D and ρ _L are the diameters of the sedimentation liquid, the target microsphere and the larger microsphere, respectively Apparent density, the choice of k value depends on the separation requirements: if the k value is set to 0.5, the separation efficiency is about 40-50% of the highest separation efficiency, and the C _V (relative standard deviation) of the obtained microspheres is lower than 3%. ; If the k value is set lower (less than 0.5), the C _V of the resulting microspheres can be further improved, but the separation efficiency will be greatly reduced; if the k value is set to 1, the separation efficiency is about the highest separation efficiency 80-95% of the obtained microspheres, the C _V of the resulting microspheres is lower than 8%; if the k value is set between 0.5-1, the efficiency of separation and the C _V of the microspheres are between the above values; in particular, k The value is set to 0.7, the number of separations is 8 times, the obtained separation efficiency is about 65% of the highest separation efficiency, the C _V of the obtained microspheres is lower than 4%, and the yield of the microspheres is 22-32%; the highest separation efficiency is based on The highest separation value that can be achieved by the separation device, in this state, the yield of microspheres is 35-50%.

In order to facilitate the sampling of microsphere samples and collect the separated microspheres without disturbing the stratification, the microsphere outlet hole 3 and the sampling microsphere outlet hole 4 are respectively provided with protruding holes relative to the side wall 2 of the barrel body. The first branch pipe 5 and the second branch pipe 6 , the valve 7 is arranged in the first branch pipe 5 and the second branch pipe 6 .

In order to prevent foreign matter from entering the barrel and prevent the solvent in the barrel from volatilizing, the mouth 13 of the barrel is provided with a dustproof cover 8 matching it.

In order to better prevent the volatilization of the solvent in the barrel, a sealing mechanism is provided at the contact between the dust-proof cover 8 and the barrel mouth 13 .

Preferably, the sealing mechanism is frosted glass 9 arranged inside the dust-proof cover 8 and outside the mouth 13 .

According to fluid mechanics, too large diameter will disturb the liquid, affect the separation effect, and reduce the yield. The ratio of the diameter d of the barrel to the height h of the barrel is d:h=1:0.5-50, preferably d:h=2.

The ratio of the distance h 1 from the microsphere exit hole 3 to the bottom of the bucket 2 and the distance h ₂ from the sampling microsphere exit hole 4 to the bottom ₂ of the bucket is h ₁ : h ₂ =1:1.5-2, preferably h ₁ :h ₂ =1:1.5, and h ₂ is between 1/10-1/3 of the barrel height h, the optimum height is about 1/4-1/3, reasonable design can optimize the separation efficiency .

Preferably, the side wall of the barrel body is provided with a loading upper limit marking line 10, a loading lower limit marking line 11 and a control marking line 12, and the loading amount in the barrel is between the loading upper limit marking line 10 and the loading lower limit marking line 11, preferably 60-80% of rated volume.

The position of the control marking line 12 is mainly determined by the density of the sedimentation liquid and the size of the target microspheres. If it is too high or too low, the yield will be reduced. The height h ₃ of the control line 12 is higher than h ₂ . The formula in the fourth chapter of the doctoral dissertation "Research on Monodisperse Functional Polymer Microspheres" deduces the specific position of the control marking line.

When using the device of the present invention to separate microspheres, the barrel body is marked with a replaceable control line translucent sticker, and according to the particle size, set the control line of different target microspheres. If the target microspheres to be separated are obtained by dispersion polymerization of 2.5 μm For microspheres, the control line is set at 2/5 of the barrel (k value is about 0.9), and the separation efficiency is 45%; 1/4 place (k value is about 0.7), separation efficiency is 22%; First, place in the barrel of the device of the present invention after the microsphere and sedimentation liquid are mixed, cover dustproof cover 8, leave standstill, when After the sedimentation interface of the target microsphere is lower than the microsphere exit hole 3, open the valve 7, discharge the microsphere with a smaller particle size, add the sedimentation liquid, repeat the process 3-20 times, when the sedimentation interface of the target microsphere reaches the control mark At line 12, follow up and detect from sampling microsphere outlet 4, until the proportion of small particle size microspheres in the discharged microspheres is less than 1%; then, add sedimentation liquid, when the sedimentation interface of the target microspheres reaches the control When the marking line is 12, follow up and detect the sampling microsphere from the microsphere exit hole 4, and discharge the target microsphere from the microsphere exit hole 3 at the same time, add the sedimentation liquid, and repeat the process 3-10 times; The proportion of smaller and larger microspheres is not higher than 1%; for specific methods, refer to Chapter 4 of Zhang Qi's doctoral thesis "Research on Monodisperse Functional Polymer Microspheres" in Tianjin University in 2010.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. It should be pointed out that for those of ordinary skill in the art, some improvements can be made without departing from the technical principle of the present invention. and modifications, these improvements and modifications should also be considered as the protection scope of the present invention.

Claims (10)

1. a microsphere segregation apparatus, including staving, described staving has drum head, staving sidewall and bung, it is characterised in that: Described staving sidewall offers microsphere respectively and portals and inspect microsphere by random samples and portal, and described sampling observation microsphere portals and is arranged on described microsphere and goes out The upside in hole and both are provided with valve, and described staving sidewall is additionally provided with control tag line, the height of described control tag line Degree h₃Can be obtained by equation below:

$h3=k\&CenterDot;x\&CenterDot;\frac{R_D^2({\mathrm{\&rho;}}_m-{\mathrm{\&rho;}}_D)}{R_L^2({\mathrm{\&rho;}}_m-{\mathrm{\&rho;}}_L)}$

Wherein, x is for loading limit level, R_DAnd R_LIt is respectively radius and the radius of bigger microsphere, the ρ of targeted microspheres_m、ρ_DAnd ρ_LPoint Not Wei sedimentation liquid, targeted microspheres and the apparent density of bigger microsphere, k is separation, and numerical value is between 0.5-1.

Microsphere segregation apparatus the most according to claim 1, it is characterised in that: described microsphere portals and inspects microsphere by random samples and portals point It is not provided with outwardly directed first arm of the most described staving sidewall and the second arm.

Microsphere segregation apparatus the most according to claim 1, it is characterised in that: it is provided with matched dust-proof at described bung Lid.

Microsphere segregation apparatus the most according to claim 3, it is characterised in that: described dust cap sets with described bung contact position There is sealing mechanism.

Microsphere segregation apparatus the most according to claim 4, it is characterised in that: described sealing mechanism is for being located at described dust cap Ground glass outside inner side and described bung.

Microsphere segregation apparatus the most according to claim 1, it is characterised in that: described staving diameter d and staving height h ratio For d:h=1:0.5-50.

Microsphere segregation apparatus the most according to claim 1, it is characterised in that: described microsphere portals to the distance of described drum head h₁Portal to distance h of described drum head with sampling observation microsphere₂Ratio be h₁:h₂=1:1.5-2, and h₂1/10-at staving height h Between 1/3.

Microsphere segregation apparatus the most according to claim 1, it is characterised in that: described staving sidewall is provided with loading upper limit mark Line, loading lower limit tag line and control tag line.

Microsphere segregation apparatus the most according to claim 1, it is characterised in that: described control tag line height h₃Higher than h₂, and Some can be set.

10. the method for utilization microsphere segregation apparatus separation microsphere as described in claim 1 to 9 is arbitrary, it is characterised in that include Following steps:

(1) microsphere and sedimentation liquid mixing are placed in described staving, stand；

(2) after the sedimentation boundary of targeted microspheres portals less than microsphere, opening the valve that microsphere portals, that discharges compared with small particle is micro- Ball, adds sedimentation liquid, repeats 3-20 time, when the sedimentation boundary of targeted microspheres arrives control tag line, portals from sampling observation microsphere Sampling observation tracing detection, until the accounting that small particle microsphere is in discharged microsphere is less than 1%；

(3) add sedimentation liquid, when targeted microspheres sedimentation boundary arrive control tag line time, from sampling observation microsphere portal 4 sampling observations with Track detects, portal discharge targeted microspheres from microsphere simultaneously, add sedimentation liquid, repeats 3-10 time, inspects by random samples and final is once separated In microsphere, less and bigger microsphere accounting is not higher than 1%.