CN108226013B - Device and method for improving particle detection precision and flux by electric field focusing - Google Patents
Device and method for improving particle detection precision and flux by electric field focusing Download PDFInfo
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- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims abstract description 12
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Abstract
The invention discloses a device and a method for improving particle detection precision and flux by utilizing electric field focusing, wherein the device comprises a PDMS micro-fluidic chip with a micro-channel, a signal amplifying circuit and a signal acquisition and processing unit, wherein the micro-channel comprises: the sample channels are respectively arranged at the front end and the rear end of the sample channel through detection channel intervals; both the two are symmetrically distributed on two sides of a straight line perpendicular to the center of the detection channel; the platinum electrode in the rear end liquid storage hole is connected with the negative electrode of each direct current power supply through a reference resistor respectively, and the platinum electrode in the front end liquid storage hole is connected with the positive electrode of each direct current power supply; the two ends of the reference resistor are connected with the input ends of the signal amplifying circuits through wires, and the output ends of the signal amplifying circuits are connected with the signal acquisition and processing units. The invention can greatly increase the detection flux and the detection precision by detecting in multiple paths in parallel in a single detection channel with multiple line widths, and effectively avoid the channel blocking phenomenon.
Description
Technical Field
The invention relates to the technical field of particle detection, in particular to a device and a method for improving particle detection precision and flux by utilizing electric field focusing.
Background
In the fields of environmental monitoring, machining, transportation, biomedicine and the like, the portable particle rapid counting device and method for accurately counting target objects (such as metal particles, bacteria, viruses, marine microorganisms and the like) in a sample have theoretical value and application prospect.
Currently available methods for particle counting include the following:
1) And (3) a photoresist method: the particle counting method is the most widely used method, and it uses the light intensity change caused by the shielding of the light by the particles to realize the detection of the particle size of the particles. The method is simple in principle, but requires an expensive and complex photoelectric conversion system, and is low in detection accuracy.
2) Inductance method: the inductance method uses the inductance change caused by particles passing through the detection microwells to achieve particle counting. The method is simple in principle, suitable for detecting various metal particles and limited in detection accuracy.
3) Capacitance method: the capacitance method is a non-contact detection method, and when particles pass through detection micropores, the change of micropore capacitance is caused, and counting can be realized by monitoring the change of micropore capacitance. The method is only suitable for counting metal particles in low-conductivity solution (such as oil liquid), but the detection of the tiny capacitance depends on a high-precision detection instrument.
4) Laser induced fluorescence detection method: the particles can carry fluorescence after laser irradiation and can be detected by a photoelectric sensor, so that particle counting is realized. The method is commonly used in the fields of blood analysis, immunology, microbiology and the like, is convenient to detect and accurate in counting, but needs pretreatment before detection, and has high cost of optical elements, complex structure and incapability of detecting very small particles.
5) Resistance pulse method (RPS): the resistance pulse method is based on that when particles flow through micropores with an electric field, voltage changes at two ends of the micropores generate detection signals, so that detection is realized; the method is simple and convenient to operate, and is the particle counting method with highest precision at present.
The microfluidic chip device is a micro device which integrates basic operations such as sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes on a micron-scale chip and automatically completes the whole analysis process. The liquid flow is controllable, the consumption of reagents and samples is extremely small, the analysis speed is improved by tens to hundreds times, and the like, so that simultaneous analysis of up to hundreds of samples can be realized in a few minutes or even shorter time, and the whole pretreatment and analysis processes of the samples are finished on line.
In recent years, the development of micro-fluidic chip processing technology is rapid, and many researches on counting micro-nano targets such as micro-nano particles, cells, bacteria and viruses on a micro-fluidic chip by utilizing RPS have been reported. The traditional microfluidic RPS counting chip is characterized in that a section of detection channel with the size slightly larger than that of particles to be detected is processed in the middle section of a straight micro channel to which a direct current electric field is applied, and the change of the size in the channel can cause the change of the distribution of the electric field of the channel, so that the local electric field intensity at the detection channel is far higher than that of the electric field at the rest part of the direct channel; when insulating particles to be detected in the solution pass through the detection channel, disturbance to an electric field in the detection channel can obviously influence voltage changes at two ends of the insulating particles, and a proper signal acquisition and processing system can acquire and output a voltage pulse signal, wherein the number of the pulse signals in the detection process is the number of the particles. Based on the above principle, different chip structures have been studied for realizing detection and counting by effectively utilizing the voltage variation.
For example, the original structure of the traditional RPS detection chip only comprises a main channel with a width of hundreds of micrometers and a detection channel with a line width slightly larger than that of particles to be detected, which is arranged in the middle of the main channel, then platinum electrodes inserted at two ends of the main channel are connected with two ends of a direct current power supply after being connected with resistors in series, and voltage signal changes at two ends of the amplified resistors are collected to realize particle counting; the improved detection chip is provided with a detection arm channel respectively in front of and behind the detection channel, voltage signals at two ends of the detection channel are directly measured by using the electrodes, and then the voltage signals are output after differential amplification, so that the detection signal-to-noise ratio and the detection precision are improved; in recent years, a high-flux counting chip adopts a structure with common liquid inlet holes distributed in a multi-channel annular mode, each channel comprises a main channel, a detection channel, a liquid storage hole and the like, the liquid inlet holes of the high-flux counting chip are connected with a power supply anode, the liquid storage holes are connected with a power supply cathode after being connected with resistors in series, the channels are used as references, the current counting chip can well eliminate system noise, and the high-flux counting is realized by controlling the channels to work sequentially.
However, it should be noted that in order to obtain a higher detection signal-to-noise ratio, the existing counting chip generally needs to ensure that the detection channels and particles are similar in size, thereby increasing the cost of channel processing. In addition, because the size of the detection port is relatively close to that of the particles, the phenomenon that the particles block the detection channel is easy to occur, so that the detection result of the particle size of the particles is error or detection is interrupted, and the accuracy and stable operation of the system are affected; finally, because the particle size is required to be similar to the detection port in the implementation of particle detection, the particle size detection range and detection flux of the channel can be greatly limited.
Disclosure of Invention
In view of the defects existing in the prior art, the invention aims to provide a device for improving the particle detection precision and flux by utilizing electric field focusing, and the device can greatly increase the detection flux and detection precision and effectively avoid the channel blocking phenomenon by carrying out multipath parallel detection in a single detection channel with multiple line widths in the detection process.
In order to achieve the above object, the present invention provides the following technical solutions:
the utility model provides an utilize electric field focus to improve device of granule detection precision and flux, this device includes PDMS micro-fluidic chip, signal amplification circuit and signal acquisition and processing unit, PDMS micro-fluidic chip is the one side that is carved with the microchannel on the PDMS substrate and encapsulates as an organic wholely with the slide glass to form the micro-fluidic chip of the microchannel that supplies the sample to await measuring to circulate, its characterized in that, the microchannel includes:
the device comprises a plurality of sample channels, wherein each sample channel is provided with a front end liquid storage hole serving as a sample inlet, and is provided with a rear end liquid storage hole serving as a waste liquid hole, and the other end of the sample channel is communicated with the detection channel; the front end and the rear end of the sample channel of each sample channel are symmetrically distributed on two sides of a symmetry axis by taking a straight line perpendicular to the center of the detection channel as the symmetry axis;
meanwhile, each front end liquid storage hole and each rear end liquid storage hole are internally inserted with a platinum electrode; the platinum electrodes in the rear end liquid storage holes are respectively connected with the cathodes of the independent direct current power supplies through a reference resistor, and the platinum electrodes in the front end liquid storage holes are connected with the anodes of the direct current power supplies; the two ends of the reference resistor are connected with the input ends of the signal amplifying circuits through wires, and the output ends of the signal amplifying circuits are connected with the signal acquisition and processing unit.
Further, the method comprises the steps of,
in the detection process, samples to be detected in N different sample channels can be detected in N different areas of the same detection channel.
Further, the method comprises the steps of,
the number of sample channels is preferably 3.
Another object of the present invention is to provide a method for particle counting based on the apparatus for improving the particle detection accuracy and flux by electric field focusing, which is characterized by comprising the steps of:
s1, dropwise adding a sample: dropwise adding PBS buffer solution into each front end liquid storage hole and each rear end liquid storage hole, and then dropwise adding a sample to be tested into each front end liquid storage hole;
s2, sample transportation: switching on each independent direct current power supply, and adjusting the liquid level height in each liquid storage hole, so that the sample to be tested in the sample injection hole flows from the front end of the sample channel to the rear end of the sample channel through the detection channel under the action of pressure until flowing into the liquid storage hole at the rear end;
s3, signal amplification acquisition analysis: and acquiring voltage pulse signals at two ends of a reference resistor when particles flow through a detection port, amplifying the acquired pulse signals through respective signal amplifying circuits, and recording and displaying corresponding detection data, namely detecting the number of sample particles by the signal acquisition and processing unit.
Compared with the prior art, the invention has the beneficial effects that:
1) When the invention is used for detection, the size of the sample particles to be detected is greatly different from that of the detection channel (for example, the sizes of the detection channel and the sample particles can be different by 10 times in the example, and the size ratio of the detection channel and the sample particles of a common detection chip is 2-3), so that the channel blockage phenomenon can be effectively avoided, and the detection stability is high;
2) The invention continuously improves the signal to noise ratio of the detection signal by increasing the ratio of the main channel (namely all channels except the detection channel) to the width of the detection port or by reducing the height of the channel, thereby improving the detection precision.
3) The invention can realize the multipath parallel independent detection of a plurality of groups of particles in the same detection channel, and has high detection flux.
Drawings
FIG. 1 is a schematic diagram of a micro-fluidic chip of a counting device according to the present invention;
fig. 2 is a system configuration diagram of the present invention.
In the figure: m, PDMS substrate, N, slide glass, A, front end liquid storage hole of first sample passageway, B, rear end liquid storage hole of first sample passageway, C, front end liquid storage hole of second sample passageway, D, rear end liquid storage hole of second sample passageway, E, front end liquid storage hole of third sample passageway, F, rear end liquid storage hole of third sample passageway, 1, front end of first sample passageway, 2, rear end of first sample passageway, 3, front end of second sample passageway, 4, rear end of second sample passageway, 5, front end of third sample passageway, 6, rear end of third sample passageway, 7, detection passageway.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Technical background: when particles flow through micropores with an electric field, the particles can generate obvious disturbance to the electric field, and meanwhile, voltages at two ends of the corresponding micropores change to generate voltage pulse signals. Based on the principle, a plurality of superimposed electric fields exist in the detection channel, a plurality of pulse signals with different amplitudes can be acquired on a plurality of amplifying circuits simultaneously when particles flow through the detection channel, because the signals generated by the channel where the particles are located are maximum, the required effective particle pulse signals can be obtained by adopting a mode of filtering out the pulse signals with the amplitudes smaller than the maximum amplitude in all the pulse signals, and the number of the effective signals can be counted to obtain the number of the particles of the detection sample.
Based on the above design background, the present invention provides a device for improving particle detection precision and flux by electric field focusing, as shown in fig. 1 (taking 3 sample channels as an example), which includes a PDMS microfluidic chip, a signal amplifying circuit, and a signal collecting and processing unit, where the PDMS microfluidic chip is a microfluidic chip that is formed by encapsulating a side of a PDMS substrate with a micro channel notched on the PDMS substrate and a glass slide into an integral body, so as to form a micro channel through which a sample to be detected flows, and the device is characterized in that the micro channel includes:
the sample channels are respectively arranged at the front end and the rear end of the sample channel at intervals through the same detection channel, one end of the front end of the sample channel is provided with a front end liquid storage hole serving as a sample injection hole, the other end of the front end of the sample channel is communicated with the detection channel, one end of the rear end of the sample channel is provided with a rear end liquid storage hole serving as a waste liquid hole, and the other end of the rear end of the sample channel is communicated with the detection channel; the front end and the rear end of the sample channel of each sample channel are symmetrically distributed on two sides of a symmetry axis by taking a straight line perpendicular to the center of the detection channel as the symmetry axis;
meanwhile, each front end liquid storage hole and each rear end liquid storage hole are internally inserted with a platinum electrode; the platinum electrodes in the rear end liquid storage holes are respectively connected with the cathodes of the 3 mutually independent direct current power supplies through a reference resistor, and the platinum electrodes in the front end liquid storage holes are connected with the anodes of the direct current power supplies; the two ends of the reference resistor are connected with the input ends of the 3 signal amplifying circuits through wires, and the output ends of the 3 signal amplifying circuits are connected with the signal acquisition and processing unit.
Further, the method comprises the steps of,
in the detection process, samples to be detected in N different sample channels can be detected in N different areas of the same detection channel.
Another object of the present invention is to provide a method for particle counting based on the apparatus for improving the particle detection accuracy and flux by electric field focusing, which is characterized by comprising the steps of:
s1, dropwise adding a sample: dropwise adding PBS buffer solution into each front end liquid storage hole and each rear end liquid storage hole, and then dropwise adding a sample to be tested into each front end liquid storage hole;
s2, sample transportation: switching on each independent direct current power supply, and adjusting the liquid level height in each liquid storage hole, so that the sample to be tested in the sample injection hole flows from the front end of the sample channel to the rear end of the sample channel through the detection channel under the action of pressure until flowing into the liquid storage hole at the rear end;
s3, signal amplification acquisition analysis: and after the collected pulse signals are amplified by respective signal amplifying circuits, recording and displaying corresponding detection data, namely the number of the detection sample particles, by the signal collecting and processing unit so as to realize detection of samples to be detected in N different sample channels in the detection process in N different areas of the same detection channel.
The detection method can identify the channel to which the counted sample belongs, namely, the sample counts of the three channels are not interfered with each other through processing the collected pulse signals. Preferably, the signal acquisition and processing unit comprises an NI acquisition card and a computer.
The following test is performed using polystyrene sample particles as an example, as shown in FIG. 2:
device parameters of this embodiment: the chip detection channels used in this embodiment have dimensions of 50×30 μm (width×length), the distance from each reservoir hole to the chip detection channel is 5mm, and the dimensions of the remaining channels are 200×20 μm (width×height); the sample to be detected is a 5 mu m polystyrene particle solution; the buffer was PBS (1×) solution; the strength of the electric field applied to the anode and the cathode of the channel is 40V/cm;
the micro-channel of the device comprises a front end liquid storage hole A of a first sample channel, a rear end liquid storage hole B of the first sample channel, a front end liquid storage hole C of a second sample channel, a rear end liquid storage hole D of the second sample channel, a front end liquid storage hole E of a third sample channel, a rear end liquid storage hole F of the third sample channel, a front end 1 of the first sample channel, a rear end 2 of the first sample channel, a front end 3 of the second sample channel, a rear end 4 of the second sample channel, a front end 5 of the third sample channel, a rear end 6 of the third sample channel and a detection channel 7; platinum electrodes are inserted into all the liquid storage holes, the platinum electrodes in the liquid storage holes at the rear end are respectively connected with the negative electrode of a direct current power supply through a reference resistor, and the platinum electrodes in the other liquid storage holes are connected with the positive electrode of the corresponding direct current power supply; two ends of each resistor R are connected in parallel with two input ends of a corresponding differential amplifier through two wires; the output end of the differential amplifier is connected to the input end of the NI data acquisition card; the NI output signal can be displayed and analyzed directly at the connected computer.
The method for counting particles based on the particle counting device comprises the following steps:
s1) dropwise adding a sample: firstly, dropwise adding 10 mu L of PBS buffer solution into each liquid storage hole, and then dropwise adding 10 mu L of PBS buffer solution and 2.5 mu L of sample to be detected into each liquid storage hole at the front end;
s2) sample transport: switching on a direct current power supply, and adjusting the liquid level height in each liquid storage hole, so that samples in each channel can flow into the rear section of each sample channel from the front section of each channel through the detection channel under the action of pressure, and then flow to the liquid storage hole at the rear end corresponding to the rear section channel;
s3) signal amplification acquisition analysis: the voltage pulse signals at two ends of each reference resistor are collected when particles flow through the detection port, the collected signals are amplified by the respective differential amplifier, and the signals are processed by the signal collection and processing system and recorded and displayed with corresponding detection data, namely the number of the particles in the detection sample.
Wherein, since the first, second and third sample channels are all applied with independent electric fields, the electric field intensity in the detection channel 7 is very high (i.e. the electric lines of force are very dense); when particles pass through the detection channel, the electric field lines can be easily disturbed, so that the voltage of the detection channel is changed, the voltage at two ends of the resistor R is further changed, a detection signal is generated, the detection signal is amplified by a differential signal of an AD620 differential amplifier and then is input to an NI acquisition card, and an output signal of the NI acquisition card can be directly displayed and analyzed on a connected computer.
Specific detection results: the real-time detection and counting results can be directly obtained through a computer, the number of particles in the sample is equal to the number of effective pulse signals, and the detection and counting results of all channels can be respectively displayed on different counting panels or distinguished by lines with different colors on the same counting panel.
In summary, in the detection process of the invention, a plurality of sample channels are used, in this example, three channels are connected with an independent power supply to focus the electric field lines in the detection channels; because the size of the detection channel is larger than that of the sample to be detected, the channel blocking phenomenon is effectively avoided, and the detection precision is improved; in addition, in the detection process, three sample channels are simultaneously detected in multiple paths in parallel in the detection channels, so that the detection and counting flux is greatly improved; and because three superimposed electric fields exist in the detection channel, three pulse signals with different amplitude values are collected on the three amplifying circuits at the same time when particles flow through the detection channel, namely, the signal generated by the channel where the particles are located is the largest, a section of comparison and filtration subroutine is added by using a filtration module such as LabVIEW program to filter out two signals with smaller amplitude values, so that the required effective particle pulse signals can be obtained, and the number of the effective signals is counted, so that the number of the particles of the detection sample can be obtained.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (3)
1. A method for particle counting using an apparatus for improving particle detection accuracy and throughput using electric field focusing, comprising the steps of:
s1, dropwise adding a sample: dropwise adding PBS buffer solution into each front end liquid storage hole and each rear end liquid storage hole, and then dropwise adding a sample to be tested into each front end liquid storage hole;
s2, sample transportation: switching on each independent direct current power supply, and adjusting the liquid level height in each liquid storage hole, so that the sample to be tested in the sample injection hole flows from the front end of the sample channel to the rear end of the sample channel through the detection channel under the action of pressure until flowing into the liquid storage hole at the rear end;
s3, signal amplification acquisition analysis: collecting voltage pulse signals at two ends of a reference resistor when particles flow through a detection port, amplifying the collected pulse signals through respective signal amplifying circuits, and recording and displaying corresponding detection data, namely detecting the number of sample particles by a signal collecting and processing unit;
the particle counting method can identify the channel to which the counted sample belongs through processing the collected pulse signals, namely the sample count of each channel is not interfered with each other;
the device for improving the particle detection precision and flux by utilizing electric field focusing comprises a PDMS micro-fluidic chip, a signal amplifying circuit and a signal acquisition and processing unit, wherein the PDMS micro-fluidic chip is formed by packaging one side of a micro-channel concavely carved on a PDMS substrate and a glass slide into a whole so as to form the micro-fluidic chip of the micro-channel for the circulation of a sample to be detected, and the micro-channel comprises:
the device comprises a plurality of sample channels, a plurality of detection channels and a plurality of detection channels, wherein each sample channel is provided with a sample channel front end and a sample channel rear end at intervals through the same detection channel, one end of the sample channel front end is provided with a front end liquid storage hole serving as a sample injection hole, the other end of the sample channel front end is communicated with the detection channel, one end of the sample channel rear end is provided with a rear end liquid storage hole serving as a waste liquid hole, the other end of the sample channel rear end is communicated with the detection channel, and the sample channel front end and the sample channel rear end of each sample channel are symmetrically distributed on two sides of a symmetry axis by taking a straight line vertical to the center of the detection channel as a symmetry axis; the sample in each channel can flow into the rear section of each sample channel through the detection channel at the front section of each channel and then flow to the rear liquid storage hole corresponding to the rear section channel;
meanwhile, each front end liquid storage hole and each rear end liquid storage hole are internally inserted with a platinum electrode; the platinum electrodes in the rear end liquid storage holes are respectively connected with the cathodes of the independent direct current power supplies through a reference resistor, and the platinum electrodes in the front end liquid storage holes are connected with the anodes of the direct current power supplies; the two ends of the reference resistor are connected with the input ends of the signal amplifying circuits through wires, and the output ends of the signal amplifying circuits are connected with the signal acquisition and processing unit.
2. The method for particle counting using an apparatus for improving particle detection accuracy and throughput using electric field focusing as claimed in claim 1, wherein:
in the detection process, samples to be detected in N different sample channels can be detected in N different areas of the same detection channel.
3. The method for particle counting using an apparatus for improving particle detection accuracy and throughput using electric field focusing as claimed in claim 1, wherein:
the number of sample channels is preferably 3.
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