Name: microfluidic platform with multiplexed electronic detection for spatial tracking of particles
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Description: Watch this Scientific Journal Video about Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles at JoVE.com

This work was supported by National Science Foundation Award No. ECCS 1610995. The authors would like to thank the Institute of Electronics and Nanotechnology and the Parker H. Petit Institute for Bioengineering and Bioscience staff for their support in using shared facilities. The authors also would like to thank Chia-Heng Chu for his help in preparing the manuscript.

코딩 전극 1. 디자인 참고 :도 1a는 미세 패턴 전극의 3 차원 구조를 나타낸다. <ol><li> 미세 유체 채널 (23)을 인코딩하기위한 네 개의 7 비트 골드 코드 세트를 설계한다. <ol><li> 이 선형 피드백 시프트 레지스터 (의 LFSR), 원시 다항식을 나타내는 각을 구축합니다. </li><li> 7 비트 m의 -sequences의 바람직한 쌍을 생성하기 위해 상기의 LFSR을 사용...

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다중 펄스 저항 센서는 이전에 미세 유체 칩 28, 29, 30, 31, 32에 포함되었다. 이러한 시스템에서, 저항 펄스 센서 중 28, 29, 30, 31 멀티플렉싱되지 않은 또는 개별 센서 <sup class="xref"...

이러한 액체 현탁 세포, 박테리아 또는 바이러스와 같은 생물학적 입자의 정확한 검출 및 분석 애플리케이션 (1, 2), (3)의 범위의 큰 관심사이다. 크기가 잘 일치, 마이크로 유체 장치는 고감도, 부드러운 시료 조작하고 잘 제어 된 미세 4, 5, 6, 7 등이 목적에 독특한 이점을 제공한다. 또한, 마이크로 유체 장치는 수동적으로 다양한 특성 8, 9, 10, 11, 12에 기초하여 생물학적 입자의 이종 집단을 분별 유체 역학 및 포스 필드의 조합을 사용하도록 설계 될 수있다. 그 장치에S는, 생성 된 입자 분포를 판독로서 사용될 수 있지만, 공간 정보는 랩 기반으로 묶어서 미세 유동 장치의 실용성을 제한 만 현미경을 통해 일반적으로 접근 할 수있다. 따라서, 그들은 마이크로 유체 장치를 조작하는 것처럼 쉽게, 입자 &#39;시공간 매핑을보고 할 수있는 통합 된 센서는 잠재적으로 저가의 모바일 샘플의 테스트에 특히 매력적이다 통합 된 랩 온어 칩 장치를 활성화 할 수 있습니다 자원 제한 설정. 박막 전극은 다양한 어플리케이션 (13), (14) 마이크로 유체 장치에 통합 센서로서 사용되어왔다. 저항 펄스 감지 (RPS)는이 전기 측정 (15)에서 직접, 강력한 민감하고 높은 처리량 감지 메커니즘을 제공으로 미세 유체 채널 작은 입자의 통합 감지에 특히 매력적이다. RPS에서는 전해액에 침지 한 쌍의 전극 간의 임피던스 변조는, 입자를 검출하기위한 수단으로 사용된다. 입자가 개구를 통과 할 때, 입자 정도의 크기, 개수 및 전류의 과도 펄스의 진폭은 각각 셀 크기 입자에 사용된다. 또한, 센서 감도 형상은 16, 17, 18, 19을 향상 시키거나 미세 유체 채널 (20)에 입자의 수직 위치를 추정하기 위해 저항 펄스 파형을 형성하기 위해 포토 리소 그래픽 해상도로 설계 될 수있다. 우리는 최근 전기 감지 (미세 유체 CODES) (21)에 의해 미세 유체 부호화 직교 검출라는 확장 가능하고 간단한 멀티 플렉스 저항 펄스 감지 기술을 도입했습니다. 미세 유체 CODES는에 의존저항 펄스 센서 네트워크 상호 다중화 가능하도록 각각 고유하게 구별 도통 변조 미세 가공 전극의 배열로 이루어진. 우리는 특히 코드 분할 다중 접속에 사용 된 디지털 코드와 유사 직교하는 전기 신호를 생성하도록 각각의 센서를 디자인했다 (22) (CDMA) 통신 네트워크, 개별 저항 펄스 센서 신호는 유일하게 하나의 출력 파형으로부터 복구 될 수 있도록 짝수 신호로부터하다면 다른 센서를 방해합니다. 이러한 방식으로, 우리의 기술은 최소한의 장치 - 시스템 레벨 복잡성을 유지하면서, 미세 유체 칩상의 다른 위치에서의 입자의 모니터링을 허용하는, 1 차원 전기 신호로 입자의 2 차원 공간 정보를 압축한다. 본 논문에서는 실험 및 전산 미세 유체 CODES 기술을 사용하는 데 필요한 방법뿐만 아니라 연구에 대한 자세한 프로토콜을 제시시뮬레이션 된 생체 시료 분석에서의 사용에서 epresentative 결과. 기술을 설명하기 위해 예로서 네 개의 다중 센서 시제품 장치로부터의 결과를 사용하여, 우리는 (2) 실험 구성의 설명을 포함하는 미세 유체 CODES 기술 마이크로 유체 장치를 생성하는 (1)에 미세 가공에 프로토콜들을 제공 할 전자 광학 및 유체 하드웨어, (3) 다른 센서로부터의 간섭 신호를 디코딩하기위한 컴퓨터 알고리즘, (4) 검출 및 미세 유체 채널의 암세포의 분석 결과. 우리는 상세한 프로토콜을 사용하여 여기에 설명 된 것을, 다른 연구자가 자신의 연구에 우리의 기술을 적용 할 수 있습니다 생각합니다.

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microfluidic platform with multiplexed electronic detection for spatial tracking of particles

생물학적 시료의 미세 가공은 일반적으로 공간적으로 관심의 생물학적 특성에 기초하여 샘플을 분별하기 위해 다양한 역장 하에서 현탁 입자의 차등 조작을 포함한다. 얻어진 공간적 분포 분석 판독으로 사용되기 위해서는, 마이크로 유체 장치는 종종 높은 비용 및 감소 된 휴대 복잡한 장비를 요구 현미경 분석을 실시한다. 이러한 한계를 해결하기 위해 마이크로 유체 칩상의 다른 위치에서의 입자의 다중 검출을위한 집적 형 전자 감지 기술을 개발했다. 미세 유체 CODES라는 우리의 기술은 1 차원 전기 신호로 2 차원 공간 정보를 압축하는 코드 분할 다중 접속과 저항 펄스 감지를 결합합니다. 본 논문에서는 미세 유동 CODES 기술의 실제 데모 검출 크기 배양 암세포 여러 미세 유체 채널을 통해 분배 제시한다. 같이고속 현미경에 의해 확인, 우리의 기술은 정확한 외부 장비의 필요없이 모든 전자 밀집 세포 집단을 분석 할 수있다. 이와 같이, 미세 유체 CODES 잠재적으로 생물학적 시료의 시점 관리 테스트에 적합 저비용 집적 랩 온어 칩 장치를 활성화 할 수있다.

네 개의 미세 유체 채널을 통해 분산 센서 4 이루어지는 미세 유체 CODES 장치는도 1b에 도시된다. (1) 복수의 셀을 병렬 및 (2) 세포를 전극에 가까이 유지 감도를 증가시키는 전극을 통해 통과 할 수 있도록이 시스템에서, 각 미세 유체 채널의 단면은 셀의 크기에 가깝도록 설계된 . 각각의 센서는 독특한 7 비트의 디지털 코드를 생성하도록 설계된다. 이 장치는 ?...

Watch this Scientific Journal Video about Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles at JoVE.com

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

우리는 다수의 미세 채널에서 입자의 검출 및 사이징 다중화, 코드 분할 다중 접속 (CDMA)과 저항 펄스 센서 (RPS)를 결합하는 집적 표면 전극 네트워크와 미세 유체 플랫폼을 보여준다.

입자의 공간 추적을위한 멀티 플렉스 전자 감지와 미세 유체 플랫폼

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

The overall goal of this procedure is to demonstrate a microfluidic platform that combines resistive pulse sensing with code division multiple access to multiplex the detection and sizing of particles in multiple microfluidic channels. This technology, named microfluidic CODES can help realize fully integrated and truly portable labware chip devices that are well suited for the point of care testing of biological samples in resources limited settings. The main advantage of this technique is that it can electronically track spatial, temporal manipulation of particles on the microfluidic chip, eliminating the need for an external instrument such as a microscope.Our technology is compatible with soft lithography and can easily be integrated into a microphotic device where particles are fractionated to provide a direct electronic readout similar to Coulter counter. To begin construction of the microfluidic device generate a set of four, seven bit gold codes. Then design four unique electrode layouts based on the gold codes using a computer aided design, or CAD software such as AutoCAD.Finally, have the Photomasked with the designed electrode layout manufactured by a Photomask supplier. Next, soak a four inch borosilicate glass wafer in a five to one piranha solution at 120 degrees Celsius for 20 minutes. After cleaning, heat the wafer on a hot plate at 200 degrees Celsius for 20 minutes to evaporate residual water.Place the clean, dry wafer in a spin coater. Apply 2 milliliters of negative photoresist solution to the wafer and spin coat at 3000 revolutions per minute for 40 seconds. Dry the spin coated wafer on a hot plate at 150 degrees Celsius for one minute.The cover the wafer with a chrome mask in the desired electrode pattern. Expose the masked photoresist surface to 365 nanometer UV light to attain 225 millijoule per centimeter squared. The bake the exposed photoresist on a hot plate at 100 degrees Celsius for one minute.Immerse the wafer in photoresist developer for 15 seconds, then wash the pattern wafer in a gentle spray of deionized water and dry the wafer under a stream of nitrogen gas. Next, place the patterned wafer in an electron beam metal evaporator. Deposit a 20 nanometer thick chrome layer and a 80 nanometer thick gold layer onto the wafer at a rate of one Angstrom per second.Then etch the underlying photoresist by ultrasonic heating the metal coated wafer in acetone for 30 minutes at 40 kilohertz and 100%amplitude. Use a dicing saw to cut the wafer into smaller pieces as needed. To begin fabricating the microfluidic channel mold, clean and dry a four inch silicon wafer in the same way as the borosilicate wafer described previously.Place the silicon wafer in a spin coater and apply four milliliters of negative photoresist solution. Spin coat the wafer at 500 rpm for 15 seconds, then at 1, 000 rpm for 15 seconds, and finally at 3, 000 rpm for 60 seconds. Set the wafer face up on an acetone soaked clean room wipe to remove residual photoresist from the back and the edges of the wafer.Bake the wafer at 65 degrees Celsius for one minute and then at 95 degrees Celsius for two minutes. Place a chrome mask pattern for the microfluidic channels on the dry wafer. Expose the photoresist to 365 nanometer UV light at 180 millijoules per centimeter squared and then bake the wafer again at 65 and 95 degrees Celsius for one and two minutes respectively.Placed the patterned wafer in a container of photoresist developer and gently shake the container for three minutes. Rinse the developed wafer in isopropanol and dry the wafer under a stream of nitrogen gas. Bake the wafer at 200 degrees Celsius for 30 minutes, then use a profilometer to check that the pattern photoresist is uniformly thick across the wafer.Place the wafer in a vacuum desiccator along with 200 microliters of trichlorosilane in an uncovered Petri Dish. Allow the wafer to sit in the desiccator with the trichlorosilane for eight hours to silanize the wafer surface. To begin assembling the device, use general purpose clean room tape to affix the silicon wafer mold in a 150 millimeter diameter Petri Dish.Add 50 grams of a 10 to one mixture of polydimethylsiloxane prepolymer to the Petri Dish and degas the mixture in a vacuum desiccator for one hour. Cure the degassed mixture at 65 degrees Celsius for at least four hours. Use a scalpel to cut out the cured PDMS layer and then peel the cured layer off the mold with tweezers.Cut the PDMS into small pieces. Punch the inlet and outlet microfluidic channel holes with a biopsy puncher. Place the PDMS layer pattern face down on clear room tape to clean the micromachined surface.Rinse the previously prepared electrode bearing glass substrate with acetone, isopropanol, and deionized water. Dry the substrate under a stream of nitrogen gas. Place the PDMS layer and the substrate in an RF plasma generator set to 100 milliwatts with the micromachine sides facing up.Activate the micromachine surfaces in oxygen plasma for 30 seconds. Next use an optical microscope to align the pattern PDMS layer with the surface electrodes. Once aligned, allow the surfaces to come into physical contact to seal the PDMS layer onto the glass substrate.It is crucial that the coating electrode pattern on glass substrate is properly aligned with the PDMS microfluidic channels. Once aligned properly, the particle interaction with the surface electrode will generate a desired code waveform for multiplexing. Bake the assembled device at 70 degrees Celsius for five minutes, glass side down.Finally solder wires to the electrode contact pads to finish device assembly. To begin the experiment, place the microfluidic device on an optical microscope stage. Connect the device reference electrode to the signal output port of a lock-in amplifier and apply a 400 kilohertz sine wave.Connect the positive and negative sensor electrodes to two independent transimpedance amplifiers. Connect both transimpedance amplifiers to the differential voltage inputs of the lock-in amplifier with the positive sensor signal to be subtracted from the negative sensor signal. Connect the demodulator output of the lock-in amplifier to a data acquisition unit.In the data acquisition software, set a sampling rate for the lock-in amplifier output of 1 Megahertz. Set up a high speed camera to optically record the operation of the device as seen under the microscope. Draw a prepared cell suspension into a syringe.Secure the sample syringe in a syringe pump and connect the syringe to the inlet channel. Direct the outlet channel to a waste container. Use the syringe pump to drive the cell suspension through the device at a constant flow rate while recording the impedance modulation signal.After experiment's completion, process the electrical data with analysis software. Compare the processed electrical signal with images from the high speed camera to create a calibration curve for cell size. A cell suspension was flowed through a microfluidic sensor device with four unique electrode patterns derived from orthogonal sensor codes.All four sensor signals were recorded from a single electrical output. The individual sensor associated with each recorded signal was identified by correlation of the recorded sensor signals with all possible codes, which produced clearly distinguishable, autocorrelation peaks. Waveforms produced by the interfering signals from simultaneous detection of cells in all four channels were resolved with an iterative algorithm.A recorded waveform was correlated with all possible codes and the largest autocorrelation peak was identified. The corresponding individual sensor signal was reconstructed and subtracted from the input waveform. The residual signal was passed to the next iteration as the input and the process continued until the residual signal produced no autocorrelation peaks.Estimated signals were refined based on an optimization algorithm seeking the best fit between the reconstructed and the original recorded waveforms using the least squares approximation. Cell location, size, and time to cross the sensor were then determined from the channel number, amplitude, duration, and relative timing of the estimated sensor signals. The procedure was validated by comparison of the electrical signals with optical measurements from the high speed camera.Once mastered, this technique is very easy to implement, because it is very simple from a hardware perspective. It has no active on-chip component. It is directly compatible with soft lithography and the signal processing relies on a simple computational algorithm.Following this protocol you can fabricate microfluidic chips with code based multiplex electrical sensors and decode electrical signals for bioanalytical measurement. This versatile, scalable, electronic sensing technology, can be readily integrated into various microfluidic devices to realize quantitative assays by spatial temporally tracking particles as they processed on the chip. After watching this vide, you should have a good understanding of how to design, fabricate, and implement a microfluidic CODES technology.

Research

JoVE Journal

Bioengineering

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단일 입자 Microfluidic 기반 유체 트랩

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연구

생체공학

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강력한, 미세 유체 세포 내 전달 플랫폼으로 휴대 뒤쪽에서

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Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

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꼬마 선 충 에 경도 이미징을 위한 미세 플랫폼

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...