Particle suspension and application thereof

Abstract

According to the particle suspension provided by the invention, the short-chain polycation peptide is used for replacing a traditional polyether or protein type dispersing agent, and meanwhile, a dynamic hydrogen bond layer is formed by the short-chain polycation peptide and ectoine, so that the overall anti-sedimentation and anti-salt capability is improved, and the particle suspension is stable in performance while low viscosity is maintained. The dispersion uniformity and optical signal stability of the fluorescent microspheres are remarkably improved, and a new particle suspension is obtained on the basis and has the particle storage characteristics of good dispersity, stable optical signals and long-term storage stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell biology, in particular to a particle suspension and its application. BACKGROUND

[0002] At present, in the cell counter, flow cytometer and fluorescence imaging system, suspended particles (such as fluorescent microspheres (Beads) standard) are widely used for equipment calibration, detection performance verification and signal quantification. However, in actual use and long-term storage, the existing fluorescent microsphere standard diluent system generally has the following problems: 1) Most fluorescent microspheres are easily attracted by van der Waals force and aggregated or settled in aqueous system, especially under low concentration or long time standing conditions, their uneven distribution will cause random error of counting results, making the detection coefficient of variation (CV%) high, thereby affecting the accuracy verification result of the instrument; 2) The existing diluent mostly uses surfactants or protein stabilizers to improve dispersibility, however, such molecules may interfere with the fluorescent signal, thereby causing attenuation and optical interference problems; 3) The diluted fluorescent microsphere suspension still faces multiple risks in long-term storage: the microspheres are slowly settled or aggregated, the supernatant concentration is reduced, causing uneven concentration; some systems contain low concentration of organic components or proteins, which are easy to become microbial breeding matrix; the fluorescent dye is attenuated by oxidation or light, causing the signal intensity to gradually decrease.

[0003] In summary, the conventional system is difficult to meet the requirements of "long-term storage stability", "optical signal stability" and "good dispersibility" at the same time, and there is a significant contradiction between dispersibility, signal retention and long-term stability: enhancing dispersibility often depends on surfactants or high molecular polymers, but it is easy to interfere with the fluorescent signal; improving signal retention requires a stable and inert system, but the dispersibility decreases; increasing the preservation and storage stability often introduces chemical additives, which may also change the optical properties of the system.

[0004] Therefore, it is urgent to develop a new type of particle suspension that maintains stable suspension of microspheres while having good fluorescent signal compatibility without introducing optical background; maintains long-term physical and chemical stability at room temperature or refrigeration conditions; and has antibacterial and antiseptic properties, suitable for long-term standardized use. SUMMARY

[0005] To solve the problems in the prior art, the present application provides a particle suspension and its application.

[0006] The first aspect of the present application provides a particle suspension, which comprises:

[0007] 0.05%-1.0% of a peptide-like substance;

[0008] 0.1%-0.5% of an antibacterial preservative;

[0009] 10mM-200mM of buffer solution;

[0010] The peptoid has the following structural unit:

[0011] [-N(CH2R1)CH2CO-] n ,

[0012] wherein R1 is a side chain containing a hydrophilic group, and n is between 5 and 10.

[0013] Preferably, the side chain containing a hydrophilic group comprises one or more of an aminoethyl group, a carboxyethyl group, a hydroxyethyl group, a guanidinopropyl group, or a hydroxypropyl group.

[0014] Preferably, the peptoid comprises one or more of the following peptoid one, peptoid two, peptoid three:

[0015] (1) Peptoid one: a 5-10 mer oligopeptoid polyamide composed of aminoethyl (-CH2CH2NH2) and hydroxyethyl (-CH2CH2OH) side chain groups, the side chains of which are combined in any ratio and order from aminoethyl and hydroxyethyl groups;

[0016] (2) Peptoid two: a 5-10 mer oligopeptoid polyamide composed of guanidinopropyl (-CH2CH2CH2-NHC(NH)NH2) and hydroxypropyl (-CH(CH3)CH2OH) side chain groups, the side chains of which are combined in any ratio and order from guanidinopropyl and hydroxypropyl groups;

[0017] (3) Peptoid three: a 5-10 mer oligopeptoid polyamide composed of two or three side chain groups from aminoethyl, carboxyethyl (-CH2CH2COOH), and hydroxyethyl, the side chain groups of which are combined in any ratio and order.

[0018] Preferably, the antibacterial preservative comprises sodium azide and / or benzyl alcohol. More preferably, the antibacterial preservative is benzyl alcohol.

[0019] Preferably, the particle suspension further comprises 0.01%-0.5% of a stabilizer. More preferably, the stabilizer is tetrahydropyrimidine or a derivative thereof.

[0020] Preferably, the buffer solution is a HEPES buffer system, a MOPS buffer solution, or a HEPES-MOPS composite buffer system. More preferably, the buffer solution has a pH value of 7.2-7.4.

[0021] The second aspect of the present application provides a method for preserving a suspended particle, comprising:

[0022] adding a suspended particle to the particle suspension as described in the first aspect of the present application, and mixing and preserving.

[0023] The third aspect of the present application provides a method for evaluating the preservation effect of suspended particles in a particle suspension, comprising:

[0024] The suspended particles are added to the particle suspension as described in the first aspect of the present application, mixed and preserved, sampled and detected, and the preservation effect of the suspended particles is evaluated according to the detection results.

[0025] Preferably, the detection index of the sampling detection includes one or more of particle suspension uniformity, particle uniformity index, and particle optical imaging dispersibility.

[0026] The fifth aspect of the present application provides a particle suspension kit, comprising the particle suspension as described in the first aspect of the present application and the suspended particle preservation method as described in the second aspect of the present application.

[0027] The sixth aspect of the present application provides the use of the particle suspension as described in the first aspect of the present application, the suspended particle preservation method as described in the second aspect of the present application, or the particle suspension kit as described in the fourth aspect of the present application in particle suspension.

[0028] Compared with the prior art, the present application has the following advantages:

[0029] The particle suspension provided by the present application replaces the traditional polyether or protein type dispersant with a short-chain polycationic peptide, and forms a dynamic hydrogen bond layer with tetrahydropyrimidine through the short-chain polycationic peptide, thereby improving the overall anti-settling and salt resistance, maintaining low viscosity, significantly improving the dispersion uniformity and optical signal stability of the fluorescent microspheres, and obtaining a new particle suspension with good dispersion, stable optical signal and long-term storage stability. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The optical microscopic imaging diagram of the particle suspension provided by the embodiment of the present application shows the particle dispersion state of Example 8 under the condition of microscopic imaging, and the particles are uniformly distributed and have consistent spacing without obvious aggregation area.

[0031] Figure 2 The suspension uniformity retention rate result diagram of the particle suspension provided by the embodiment of the present application;

[0032] Figure 3 The sedimentation uniformity index result diagram of the particle suspension provided by the embodiment of the present application;

[0033] Figure 4 The aggregation area proportion result diagram of the particle suspension provided by the embodiment of the present application. DETAILED DESCRIPTION

[0034] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be clearly and completely described below with reference to the drawings in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0035] A particle suspension, comprising:

[0036] 0.05%-1.0% of a peptoid;

[0037] 0.1%-0.5% of an antimicrobial preservative;

[0038] 10mM-200mM of a buffer;

[0039] The peptoid has the following structural unit:

[0040] [-N(CH2R1)CH2CO-] n ,

[0041] wherein R1 is a side chain containing a hydrophilic group, and n is between 5 and 10.

[0042] In some embodiments, the side chain containing a hydrophilic group comprises one or more of an aminoethyl group, a carboxyethyl group, a hydroxyethyl group, a guanidinopropyl group, or a hydroxypropyl group.

[0043] In some embodiments, the peptoid comprises one or more of the following peptoid one, peptoid two, peptoid three:

[0044] (1) Peptoid one: a 5-10 mer oligopeptoid polyamide composed of aminoethyl (-CH2CH2NH2) and hydroxyethyl (-CH2CH2OH) side chain groups, the side chains of which are combined in any ratio and order from aminoethyl and hydroxyethyl groups;

[0045] (2) Peptoid two: a 5-10 mer oligopeptoid polyamide composed of guanidinopropyl (-CH2CH2CH2-NHC(NH)NH2) and hydroxypropyl (-CH(CH3)CH2OH) side chain groups, the side chains of which are combined in any ratio and order from guanidinopropyl and hydroxypropyl groups;

[0046] (3) Peptoid three: a 5-10 mer oligopeptoid polyamide composed of two or three side chain groups from aminoethyl, carboxyethyl (-CH2CH2COOH), and hydroxyethyl, the side chain groups of which are combined in any ratio and order.

[0047] In some embodiments, the antimicrobial preservative comprises sodium azide and / or benzyl alcohol. In some preferred embodiments, the antimicrobial preservative is benzyl alcohol.

[0048] In some embodiments, the fluorescent microsphere preservative solution further comprises 0.05%-0.5% of a stabilizer. In some preferred embodiments, the stabilizer is tetrahydropyrimidine or a derivative thereof.

[0049] In some embodiments, the buffer is a HEPES buffer system, a MOPS buffer or a HEPES-MOPS composite buffer system. In some preferred embodiments, the buffer has a pH value of 7.2-7.4.

[0050] Examples

[0051]

[0052] In which, the peptide-like is conceived and the sequence is as follows: short-chain polycationic peptide-like (5-10 mer), consisting of 5-10 N-substituted glycine units, having a molecular weight of about 0.8-2.0 kDa, belonging to the oligomeric peptide mimetic, synthesized and sub-packed by Kings River Company. The following is the peptide-like 1:

[0053]

[0054] In addition, it needs to be explained that the antibacterial preservative in the above examples selects benzyl alcohol as an example, and those skilled in the art know that the replacement or superposition of functionally similar sodium azide can also be used as the antibacterial preservative in the present application. Similarly, the buffer in the above examples selects the HEPES buffer system as an example, and those skilled in the art know that the replacement or superposition of functionally similar MOPS buffer system can also be used as the buffer in the present application.

[0055] Application Example 1: Comparison and verification of different particle suspensions

[0056] Experimental design

[0057] A suspension with a concentration of 1×10 6 beads / mL was prepared using standard 10 μm polystyrene microspheres (Beads), and the particle suspensions of Examples 1-3 and Comparative Example 1 were dispersed, respectively.

[0058] Detection verification

[0059] (1) Suspension uniformity

[0060] 1) Sample preparation: Take 10 mL of the particle suspensions of Examples 1-3 and Comparative Example 1, respectively. Add standard 10 μm polystyrene microspheres (Beads) to each, and adjust to a final concentration of 1×10 6 beads / mL. Mix well using vortex oscillation for 10 s, and immediately start timing.

[0061] 2) Detection time points: Set 0h, 24h, 48h as three determination time points. Lightly invert the test tube 3 times before each time point to avoid false uniformity caused by strong shaking.

[0062] 3) Determination method: Use spectrophotometer or enzyme marker (wavelength 600nm) to determine the optical density (OD600) of each sample. Take 200μL of each sample and place it into a transparent 96-well plate for determination, with 3 parallel holes in each group. Immediately seal the film after each measurement to avoid evaporation.

[0063] 4) Data processing: Calculate the average OD600 value of each group at each time point.

[0064] Take OD6000 at 0h as 100% uniformity reference to calculate the relative turbidity retention rate of 24h and 48h:

[0065]

[0066] If the retention rate decreases by more than 10%, it indicates that the particles start to settle.

[0067] 5) Evaluation criteria:

[0068] Good uniformity: 48h retention rate ≥85%;

[0069] Good uniformity: 48h retention rate 80-85%;

[0070] Poor uniformity: 48h retention rate <80%.

[0071] Experimental results

[0072]

[0073] Result analysis

[0074] This experiment compared the effects of different diluent systems on the uniformity of particle suspension. The signal change trend of each system at 0h, 24h and 48h is consistent overall, but the retention rate is significantly different.

[0075] Among them, Comparative Example 1 performed the worst, with a 48h suspension uniformity retention rate of only 53%, and a 24h suspension uniformity retention rate of only 71%, indicating that the conventional buffer system cannot effectively maintain the uniformity of particle suspension and is not suitable for the preservation of particle suspension.

[0076] The suspension uniformity retention rate of Example 1 is 88% at 24h and 77% at 48h, and there is a certain degree of particle sedimentation, which does not reach the expectation, but is obviously better than Comparative Example 1, indicating that the particle suspension provided by Example 1 can slow down the particle sedimentation to a certain extent, and the 24h suspension uniformity retention rate can basically guarantee the short-time storage effect of the particle suspension, and has a certain degree of anti-aggregation and anti-settling ability.

[0077] Example 2 performs best, with a suspension uniformity retention rate of 92% at 24h and 86% at 48h, which is higher than the requirement of “uniformity is excellent” (≥85%) in the evaluation standard, indicating that the particle suspension provided by Example 2 can effectively inhibit particle sedimentation within 48h, and has stable anti-aggregation and anti-settling ability.

[0078] Example 3 has a suspension uniformity retention rate of 82% at 48h, which is in the interval of 80-85%, and is evaluated as “uniformity is good”. The suspension uniformity retention rate at 24h is 90%, and the decrease is small, indicating that although the overall stability of the particle suspension provided by Example 3 is slightly inferior to that of Example 2, it still maintains reliable uniformity and has good anti-aggregation and anti-settling ability.

[0079] In summary, Example 2 > Example 3 > Example 1 > Comparative Example 1. Among them, Example 2 has reached the excellent standard and is the most suitable formula for long-term microsphere suspension, which can be used as the core system for subsequent optimization and application.

[0080] Application Example 2: Verification of the effect of stabilizer on the suspension uniformity of the particle suspension

[0081] To further optimize the suspension uniformity of the particle suspension, the present application takes Example 2 as an example, and adds the stabilizer ectoine to improve the optical and structural stability of the particle suspension:

[0082]

[0083] The experimental design and detection verification are the same as the above-mentioned application example 1.

[0084] Experimental results

[0085]

[0086] Result analysis

[0087] In application example 2, the amount of ectoine (Example 4-6) is gradually increased in the basic system (Example 2) to evaluate the effect of the stabilizer on the uniformity of the particle suspension.

[0088] From the experimental data, the suspension uniformity retention rate of Example 4 at 24h and 48h is 93% and 85% respectively, which is similar to Example 2 without adding tetrahydropyrimidine, and both reach the evaluation standard of "uniformity is excellent" (≥85%), while the suspension uniformity retention rate of Example 5 at 24h reaches 95% and at 48h reaches 88%, both of which are better than Example 2 without adding tetrahydropyrimidine, indicating that the addition of low-concentration tetrahydropyrimidine not only does not cause additional burden to the system stability, but also further improves the particle suspension performance of the particle suspension.

[0089] When the addition amount of tetrahydropyrimidine is increased to 0.5% (Example 6), its retention rate at 24h and 48h decreases to 90% and 82% respectively, which belongs to "uniformity is good". Although the suspension ability is still good, the dispersion uniformity is slightly lower than that of Examples 4 and 5. The results show that at a higher concentration, the hydration structure enhanced by tetrahydropyrimidine may cause a slight increase in the viscosity of the system, resulting in a certain degree of sedimentation of the particles in the later stage (24-48h), but it does not affect the overall uniformity evaluation grade.

[0090] Comprehensive analysis, the addition of low-concentration tetrahydropyrimidine can maintain the optical transparency and structural stability of the suspended particles in the particle suspension, while not affecting the suspension performance of the particles. While the addition of higher concentration of tetrahydropyrimidine does not lead to system instability, but does not bring further advantages. That is, the addition of low-concentration tetrahydropyrimidine can improve the optical transparency and structural stability of the particle suspension system without changing the uniformity grade of the system.

[0091] Application Example 3: Verification of the particle preservation effect of different peptide-like peptides on particle suspension

[0092] To further optimize the particle preservation effect of the particle suspension, the present application takes Example 5 as an example, and designs two short-chain polycation peptides (5-10mer) with different structural characteristics to replace peptide-like peptide one for comparison and verification:

[0093]

[0094] Among them, the sequence, design principle and expected effect of peptide-like peptide two and peptide-like peptide three are as follows:

[0095]

[0096] To verify the particle preservation effect of the particle suspension, the sedimentation rate detection method and the optical imaging dispersion detection method are added on the basis of the suspension uniformity detection, and the specific experimental design and detection verification are as follows:

[0097] (1) Sedimentation rate detection method

[0098] 1) Sample preparation: Take 10 mL of each particle suspension of Example 5, 7, 8 and Comparative Example 1, respectively. Add standard 10 μm polystyrene microspheres (Beads) to adjust the final concentration to 1 x 10 6 particles / mL. Vortex for 10 s and immediately place in a transparent centrifuge tube.

[0099] 2) Test conditions: Use a benchtop low speed centrifuge with 500 x g for 2 min. Do not shake or agitate the test tube before and after centrifugation. Test at room temperature (25 ± 2 °C).

[0100] 3) Observation and recording:

[0101] Before centrifugation: Mark three positions (1 cm, 3 cm, 5 cm from the bottom) on the tube wall. Take 200 μL of the suspension from each position using a pipette.

[0102] After centrifugation: Repeat the above sampling positions.

[0103] Each sample is tested in triplicate.

[0104] 4) Test method:

[0105] Determine the OD600 or count the number of particles on a cell counter for each sample. Record the particle concentration or optical density of the upper, middle and lower layers.

[0106] 5) Data processing:

[0107] Calculate the ratio of particle distribution before and after centrifugation, and define the uniformity index (UI):

[0108]

[0109] wherein and are the particle concentrations of the upper and lower layers, respectively.

[0110] A UI close to 1 indicates uniform particle distribution, and the smaller the UI, the more obvious the sedimentation.

[0111] 6) Evaluation criteria:

[0112] Excellent anti-settling performance: UI > 0.9 (difference in concentration between upper and lower layers < 10%).

[0113] Good anti-settling performance: 0.8 < UI < 0.9.

[0114] Poor anti-settling performance: UI < 0.8.

[0115] (2) Optical imaging dispersibility test method

[0116] 1) Sample preparation: Take 2 mL of each particle suspension from Example 5, 7, 8 and Comparative Example 1, respectively. Add standard 10 pm polystyrene microspheres (Beads) to adjust the final concentration to 5 x 10 5 Immediately after vortex mixing for 5 s, take samples.

[0117] 2) Tabletting method:

[0118] Take 10 pL of the suspension and drop it onto a clean glass slide, cover with a cover glass. Let it stand for 2 min, allowing the particles to distribute naturally, without drying. If fluorescent Beads are used, they can be observed directly under a fluorescence microscope; non-fluorescent Beads can be imaged using bright field.

[0119] 3) Microscopy imaging conditions:

[0120] Microscope magnification: 20x or 40x objective; imaging device: inverted microscope + digital camera; take images of 5 different fields at random for each sample; use the same exposure and contrast settings for all samples to ensure comparability.

[0121] 4) Image analysis:

[0122] Use ImageJ or similar image analysis software. Perform the following analysis steps for each image: particle recognition and counting (Analyze Particles function); calculate the average inter-particle distance and the Cluster Area Ratio (CAR):

[0123]

[0124] Calculate the average CAR value for 5 fields.

[0125] 5) Evaluation criteria:

[0126]

[0127] Experimental results

[0128] (1) Suspension homogeneity retention rate (OD600 stability)

[0129]

[0130] (2) Sedimentation rate detection (Upper / Lower layer concentration ratio UI)

[0131]

[0132] After low-speed centrifugation, the particles in Comparative Example 1 were clearly sedimented (UI = 0.62 ± 0.03), while Example 8 maintained a nearly uniform distribution (UI = 0.95 ± 0.01).

[0133] (3) Optical imaging dispersibility (concentration area ratio CAR)

[0134]

[0135] Result analysis

[0136] (1) The suspension uniformity retention rates (OD600 stability) of Examples 5, 7 and 8 at 24 h are all above 90%, and still above 85% at 48 h, while the suspension uniformity retention rate of Comparative Example 1 at 24 h is only about 70%, and even decreases to about 50% at 48 h, indicating that the particle suspensions provided by Examples 5, 7 and 8 have obvious effects on the particle suspension stability preservation.

[0137] (2) The uniformity indexes (UI) of Examples 5, 7, 8 and Comparative Example 1 before centrifugation are all above 95%, but that of Comparative Example 1 decreases to about 60% after centrifugation, while those of Examples 5, 7 and 8 can still be maintained at about 90%, indicating that the particle suspensions provided by the present application have excellent anti-settling performance, and can effectively prevent the suspended particles of the particle suspensions from settling and stratifying.

[0138] (3) The concentration area ratios CAR of Examples 5, 7 and 8 are between 3% and 7%, while that of Comparative Example 1 is close to 15%, indicating that the optical imaging dispersibility of the particle suspensions provided by the present application is excellent, and is obviously superior to that of Comparative Example 1, and can effectively prevent the suspended particles of the particle suspensions from focusing and grouping.

[0139] (4) The microscopic imaging results are consistent with the turbidity and sedimentation tests, and Examples 5, 7 and 8 can all significantly improve the suspension stability and dispersibility of the particles in the suspensions, and among them, Example 8 (containing a hydroxyl-amino double-substituted structure) performs best in the three indexes, and has potential application value as a standard diluent or performance verification liquid for a counting instrument.

[0140] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: they can still modify the technical solutions recorded in the foregoing examples, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A particle suspension, characterized in that, comprising: 0.05%-1.0% of a peptoid; 0.1%-0.5% of an antibacterial preservative; 10mM-200mM of a buffer; the peptoid has the following structural unit: [-N(CH2R1)CH2CO-] n , wherein R1 is a side chain containing a hydrophilic group, and n is between 5-10.

2. The particle suspension of claim 1, wherein, the side chain containing a hydrophilic group includes one or more of an aminoethyl group, a carboxyethyl group, a hydroxyethyl group, a guanidinopropyl group, or a hydroxypropyl group.

3. The particle suspension of claim 2, wherein the peptoid includes one or more of the following peptoid one, peptoid two, and peptoid three: (1) peptoid one: a 5-10 mer oligopeptoid polyamide composed of aminoethyl (-CH2CH2NH2) and hydroxyethyl (-CH2CH2OH) side chain groups, the side chains of which are combined in any ratio and order with aminoethyl and hydroxyethyl groups; (2) peptoid two: a 5-10 mer oligopeptoid polyamide composed of guanidinopropyl (-CH2CH2CH2-NHC(NH)NH2) and hydroxypropyl (-CH(CH3)CH2OH) side chain groups, the side chains of which are combined in any ratio and order with guanidinopropyl and hydroxypropyl groups; (3) peptoid three: a 5-10 mer oligopeptoid polyamide composed of two or three of aminoethyl, carboxyethyl (-CH2CH2COOH), and hydroxyethyl side chain groups, the side chain groups of which are combined in any ratio and order.

4. The particle suspension of claim 1, wherein the antibacterial preservative includes sodium azide and / or benzyl alcohol.

5. The particle suspension of claim 1, wherein the particle suspension further includes 0.01%-0.5% of a stabilizer.

6. The particle suspension of claim 1, wherein the buffer is a HEPES buffer system, a MOPS buffer, or a HEPES-MOPS composite buffer system.

7. A method for preserving suspended particles, characterized by, comprising: adding the suspended particles to the particle suspension of any one of claims 1-6, mixing, and storing.

8. A method for evaluating the particle suspension preserving effect of a particle suspension, characterized by, comprising: adding the suspended particles to the particle suspension of any one of claims 1-6, mixing, storing, sampling, and evaluating the particle suspension effect according to the results of the sampling.

9. A particle suspension kit, characterized in that, the particle suspension of claim 1 and the method of claim 7.

10. Use of the particle suspension of claim 1, the method of claim 7, or the particle suspension kit of claim 9 in particle suspension.

Images