JP2008188592A - Particle screening method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve such shortcomings that for example, the use of a few units of high-output lasers and a high-voltage generator for screening, in the existing chromosome sorter, a diversion model of a cell sorter, results in the scaling up of a screening device and the requirement of large power in actual operation, and further, that a fluorescent coloring matter is needed for staining, and the coloring matter needs to be excited using the high-output laser, but with the consequent unavoidable suffering of chemical damage to and also, of mechanical damage to the molecules of a specimen by a screening process at applied high voltage. <P>SOLUTION: This method employs a microfluid device chip a few centimeters square and a device which generates an optional ambipolar waveform with voltage of a few volts, as constituents. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to the fields of clinical medicine and colloid interface science, and relates to a cell sorter, a chromosomal sorter, a vesicle sorter, a colloidal particle sorter, and the like.

  Sieve has long been used as a method of sorting particles according to size. However, in order to apply this method to the selection of biomolecules, it is necessary to produce a molecular size sieve with high accuracy. Submicron-scale molecular sieves that have made full use of recent semiconductor microfabrication technology have been produced at some research institutions. However, it is far from practical use due to various problems such as clogging by molecules, difficulty in recovery after sorting, and the need for large-scale research facilities such as clean rooms.

  The sorter for biomolecules already on the market has the basic principle of staining biomolecules with a fluorescent dye, exciting it with a laser, and selecting according to the fluorescence intensity emitted from the dye (see Non-Patent Document 1). .

J.W.Gray et al., "Science" 238, 323-329 (1987)

  For example, an existing chromosome sorter that uses a cell sorter basically uses several high-power lasers and a high-voltage generator for sorting. Therefore, the sorting device is large-scale, and high power is required for actual operation. It was. In addition, it requires staining with a fluorescent dye, and because the dye is excited by a high-power laser, the sample molecule is chemically damaged and mechanically damaged by a sorting process by applying a high voltage. Had.

  In order to solve the above problems, the present invention basically comprises a microfluidic device chip of several centimeters square and an arbitrary voltage waveform generator. The applied voltage is a few volts and is a bipolar voltage.

  Therefore, the configuration is compact, energy-saving operation is possible, basically no staining with fluorescent dye is required, and since bipolar voltage is applied, Joule heat like electrophoresis does not occur. .

The effects of the present invention are as follows.
1. The entire system consists of a microfluidic device chip of several centimeters square and a notebook computer that generates an arbitrary voltage waveform, so it is space-saving (compact) and can be operated with less than 1 / 100th the power consumption of conventional sorters. is there.
2. There is almost no mechanical and chemical damage because basically no staining with fluorescent dyes is required.
3. Sorting is possible by applying a voltage of about several volts, and since bipolar voltages are applied, Joule heat like electrophoresis does not occur.
4). By treating the surface of the microfluidic device chip and controlling the interaction force between the biomolecules or colloidal particles and the chip surface, such as electrostatic attractive force, chemical / physical adsorption force, and frictional force, more precise and in a short time Sorting becomes possible.
5. Since cells and blood cells change their electrical response characteristics and surface condition depending on their life and death, they can be sorted and discriminated by life and death by combining with 4.

  FIG. 1 is a plot of the vibration amplitudes of chromosomes of different sizes in an alternating electric field versus the alternating voltage frequency. As can be seen from the figure, the oscillation amplitude decreases with increasing AC voltage frequency for any size chromosome, and larger size chromosomes also at lower frequency AC fields compared to smaller chromosomes. Obviously it becomes impossible to respond.

  By utilizing the vibration behavior characteristics of chromosomes of different sizes in such an alternating electric field and using the voltage modulation waveform shown in FIG. 2 for selection, a high-speed and ultra-small chromosome sorter can be realized.

  An example is shown in FIG. Under these conditions (frequency: 1 Hz, applied voltage: 10 Vpp, rise time: 250 milliseconds, fall time: 40 milliseconds), small size chromosomes overtake large size chromosomes as shown in the figure. The situation is observed. In this case, the chromosome of 3 μm size showed a movement of 5 μm per pulse, and the chromosome of 9 μm size showed a movement of 0.2 μm per pulse.

  FIG. 4 is a cross-sectional view of a microfluidic device having a size of several centimeters. A microelectrode is produced on a glass substrate, a very shallow pot is formed with a polymer, and charged particles as a sample are accommodated in the pot. The device is inexpensive and disposable.

  FIG. 5 is an overview of the particle sorting apparatus according to the present invention, which includes the microfluidic device chip and a notebook computer that generates an arbitrary voltage waveform. In an alternating electric field, the mobility of small chromosomes is greater than the mobility of large chromosomes, so they move farther away and can be sorted by size. This is moved to a collection pot by a DC electric field and collected. Therefore, a large chromosome is recovered in the pot A close to the sample introduction port, and a small chromosome is recovered in the distant pot C.

  Thus, a chromosome sorter can be realized with a compact and simple apparatus configuration. In principle, this sorting technique is applicable to sorting colloidal particles as well as other biomolecules such as cells and DNA, since any type can be used as long as it is a charged substance. Already, operation experiments with colloidal particles of different sizes have been carried out to prove the principle.

The figure which plotted the vibration amplitude of the chromosome of a different size in an alternating current electric field with respect to alternating current voltage frequency. An example of a voltage modulation waveform. Here is an asymmetric bipolar waveform. Characteristics of vibration behavior of chromosomes of different sizes. Sectional drawing of a microfluidic device. The whole conceptual diagram of the particle sorter concerning the invention in this application.

Claims (2)

In a method of sorting charged particles having a size of 1 mm or less, the particles having different sizes are accommodated in a microfluidic device chip, and a voltage modulation waveform having an asymmetric bipolar waveform is applied to the particles, thereby A particle sorting method characterized by sorting. A particle sorting apparatus comprising: a microfluidic device chip that stores charged particles having different sizes of 1 mm or less; and an application unit that applies a voltage modulation waveform having an asymmetric bipolar waveform to the particles.