CN-121975603-A - High-flux biological particle separation system and method based on microfluidic chip

CN121975603ACN 121975603 ACN121975603 ACN 121975603ACN-121975603-A

Abstract

The invention discloses a high-flux biological particle separation system based on a microfluidic chip, which comprises a microfluidic BAW chip, a microfluidic cascade chip and a plurality of DLD separation units, wherein an extracellular vesicle collecting channel is configured in a separation outlet area in the microfluidic BAW chip, a radiation component comprises a first piezoelectric transducer and a second piezoelectric transducer which are respectively used for providing high-frequency bulk acoustic waves and low-frequency bulk acoustic waves, a liquid inlet channel of the microfluidic cascade chip is connected with the extracellular vesicle collecting channel, a fluid distribution area is provided with a plurality of parallel branch channels, and the DLD separation units are connected with the parallel branch channels in a one-to-one correspondence manner. Through reasonable configuration of the multi-stage separation structure, multi-target separation of biological particles with different dimensions in a blood sample can be realized.

Inventors

YE HAIHANG
HUANG PIJIANG
PAN TINGRUI
Lan Huaize
LIU BINYAO

Assignees

中国科学技术大学苏州高等研究院
中国科学技术大学

Dates

Publication Date: 20260505
Application Date: 20260210

Claims (10)

1. A microfluidic chip-based high-throughput biological particle separation system, comprising: the microfluidic BAW chip comprises a chip body and a radiation component; The chip body is provided with a first inlet area, a main separation channel and a separation outlet area which are sequentially connected, wherein the first inlet area is provided with a first sheath inflow port channel and a first sample inlet channel, the separation outlet area is provided with a plurality of outlet channels, and at least one of the outlet channels is an extracellular vesicle collecting channel; The radiation component comprises a first piezoelectric transducer and a second piezoelectric transducer, wherein the first piezoelectric transducer is used for providing high-frequency bulk acoustic waves acting on the upstream area of the main separation channel, and the second piezoelectric transducer is used for providing low-frequency bulk acoustic waves acting on the downstream area of the main separation channel; the microfluidic cascade chip comprises a liquid inlet channel and a fluid distribution area, wherein the liquid inlet channel is connected with an extracellular vesicle collecting channel in a separation outlet area, and the fluid distribution area is provided with a plurality of parallel branch channels; The DLD separation units are connected with the parallel branch channels in a one-to-one correspondence manner, and are respectively independent microfluidic DLD chips or functional units integrated in the same microfluidic DLD chip.
2. The high-throughput biological particle separation system of claim 1, wherein the main separation channel has a channel width of 1/2 wavelength of low frequency bulk acoustic waves and a channel aspect ratio of 1:3-2:3.
3. The high-flux biological particle separation system of claim 1, wherein the first piezoelectric transducer and the second piezoelectric transducer are arranged at intervals along the fluid flow direction on the side wall of the chip body, the first piezoelectric transducer and the second piezoelectric transducer are piezoelectric ceramic transducers, the thickness of the first piezoelectric transducer is 0.4 mm-0.5 mm, and the thickness of the second piezoelectric transducer is 1 mm-1.2 mm.
4. The high throughput biological particle separation system of claim 1, wherein in said microfluidic BAW chip, first sample inlet channels are symmetrically distributed on both sides of a first sheath inlet channel; The plurality of outlet channels of the separation outlet zone comprise a first central outlet channel which is coaxially arranged with the main separation channel and six first lateral outlet channels which are symmetrically arranged at two sides of the first central channel, and the two first lateral outlet channels at the outermost side are extracellular vesicle collecting channels; the microfluidic cascade chip further comprises a buffer zone arranged in the liquid inlet channel and the fluid distribution zone.
5. The high throughput biological particle separation system of claim 4, wherein in said microfluidic BAW chip, the channel width of the main separation channel is 375±25 μm and the channel depth is 200±25 μm.
6. The high throughput biological particle separation system of claim 1, wherein said DLD separation unit comprises a second inlet zone, a first separation outlet zone, a second separation zone, and a second separation outlet zone connected in sequence, wherein said second inlet zone is configured with a second sheath flow inlet channel and a second sample inlet channel connected in one-to-one correspondence with parallel branch channels, wherein a first critical separation dimension of said first separation zone is greater than a second critical separation dimension of said second separation zone, wherein said first separation outlet zone and said second separation outlet zone are each configured with at least two outlet channels, and wherein one of said outlet channels of said first separation outlet zone is connected to said second separation zone.
7. The high throughput biological particle separation system of claim 6, wherein in said DLD separation unit, second sheath flow inlet channels are symmetrically disposed on either side of second sample inlet channels; The outlet channel in the primary separation outlet zone comprises a second central outlet channel and second lateral outlet channels symmetrically distributed on two sides of the second central outlet channel, and the second central outlet channel is connected with the secondary separation zone; The outlet channel in the secondary separation outlet zone comprises a third central outlet channel and third lateral outlet channels symmetrically distributed on two sides of the third central outlet channel, and the third lateral outlet channels are respectively used for collecting exosomes and microvesicles in extracellular vesicles; the critical separation size of the primary separation area is 2um, and the critical separation size of the secondary separation area is 500nm.
8. The high-throughput biological particle separation system of any one of claims 1 to 7, wherein the plurality of DLD separation units have the same structural parameters, the plurality of parallel branch channels have the same structural parameters, and the number of DLD separation units and parallel branch channels are the same.
9. A high-throughput bio-particle separation method based on a microfluidic chip, characterized in that it is based on the high-throughput bio-particle separation system according to any one of claims 1 to 8, comprising: Introducing a sample flow into a microfluidic BAW chip, and generating high-frequency bulk acoustic waves respectively acting on an upstream area of a main separation channel and low-frequency bulk acoustic waves respectively acting on a downstream area of the main separation channel by applying driving signals with different frequencies and amplitudes to a first piezoelectric transducer and a second piezoelectric transducer, so that biological particles with different preset size ranges are pre-focused on two sides of a channel wall in the upstream area of the main separation channel, and then differential acoustophoretic migration behaviors are generated in the downstream area of the main separation channel and separation is realized; sample flows collected by an extracellular vesicle collecting channel in the microfluidic BAW chip enter the microfluidic cascade chip and are distributed to DLD separation units; In the DLD separation unit, biological particles with the dimension larger than the first critical separation dimension in the sample flow are discharged in a first separation outlet area after being separated in a first separation area, and microvesicles with the dimension larger than the second critical separation dimension and exosomes with the dimension smaller than the second critical separation dimension in the sample flow entering a second separation area are respectively collected in the second separation outlet area after being separated.
10. The method for separating high-flux biological particles according to claim 9, wherein the resonance frequency of the high-frequency bulk acoustic wave is 3.6-4.3 MHz, the voltage amplitude is 12-15V, the resonance frequency of the low-frequency bulk acoustic wave is 1.7-2.2 MHz, and the voltage amplitude is 3-5V; When the outlet channel of the separation outlet zone has seven outlet channels, the outlet channels are respectively used for collecting white blood cells, red blood cells, platelets and extracellular vesicles in blood from the center to the two sides.

Description

High-flux biological particle separation system and method based on microfluidic chip Technical Field The invention belongs to the technical field of microfluidic technology and biological particle treatment, and particularly relates to a high-flux biological particle separation system and method based on a microfluidic chip. Background Along with the development of biomedical research and accurate medical treatment, the requirements for efficiently separating and purifying various biological particles in complex biological samples such as blood and the like are increasingly urgent. Blood samples typically contain blood cell components of different sizes, such as white blood cells, red blood cells, platelets, etc., together with extracellular vesicles (Extracellular Vesicles, EVs) with sizes distributed in the nanoscale range. The biological particles have remarkable differences in size range, physical properties and biological functions, and the cooperative existence of the biological particles makes the separation process face technical challenges of various separation targets, large scale span, complex sample components and the like. At present, the microfluidic technology is widely applied to the fields of blood cell analysis and EVs separation by virtue of the accurate regulation and control capability of the microfluidic technology on fluid behaviors under microscale conditions. The deterministic lateral displacement (DETERMINISTIC LATERAL DISPLACEMENT, DLD) technology is used as a typical passive microfluidic separation method, and a regular micro-column array structure is arranged in a microfluidic channel to reconstruct a fluid flow field, so that fine separation based on particle size difference is realized. The technology has certain advantages in the aspect of high-resolution separation of nano-scale particles and EVs. However, the separation performance of DLD technology is highly dependent on the geometry of the micropillar array, and once its separation critical dimensions are determined, it is difficult to accommodate the needs of a variety of separation targets. In an actual blood sample, there is a significant difference in the size distribution of the different blood cells. For DLD arrays, each time a cell of a different critical size is isolated, it generally means that the corresponding micro-column array structure needs to be redesigned, and when multiple sizes of blood cells need to be fractionated, multiple sets of micro-column array structures of different parameters often need to be cascaded. This not only adds significant structural complexity to the chip, but also introduces additional flow resistance, which increases the overall pressure drop across the system, further limiting the achievable throughput. Furthermore, DLD technology is typically required to operate at lower flow rates in actual operation, and its overall throughput is very limited, making it difficult to meet the application requirements of high throughput separations. The acoustic separation technology introduces a sound field into the microfluidic channel, applies acoustic radiation force to suspended particles, can realize non-contact control of particles with different sizes and physical properties, and overcomes the limitation that DLD technology is difficult to separate various target particles to a certain extent. However, in high purity separation applications for nanoscale EVs, acoustic separation techniques still face flux-limiting problems. This is mainly because according to the theory of acoustic radiation force, particles with smaller size are usually excited by using a higher frequency acoustic wave, while the existing high frequency acoustic wave is mostly realized by exciting surface acoustic wave by using a piezoelectric material such as Lithium Niobate (LN), and the acoustic energy is mainly limited to the surface of a microfluidic channel, so that it is difficult to efficiently couple to the whole fluid region. The preparation cost of the acoustic chip is high and complex, so that the significant improvement of the flux is difficult to realize in practical application in a simple parallel connection mode. The method for realizing separation by using lead zirconate titanate (PZT) and other materials for bulk acoustic wave excitation can improve the separation flux by one order of magnitude, but has poor separation effect on particles below the nanometer level. In conclusion, the existing particle separation technology cannot realize multistage separation of blood cells and high-efficiency purification of EVs at the same time aiming at complex biological samples, and can give consideration to high throughput processing capability. Disclosure of Invention Based on the problems, the invention provides a high-flux biological particle separation system and a high-flux biological particle separation method based on a microfluidic chip, which combine a bulk acoustic wave (Bulk Acoustic Wave, BAW) sepa