Name: microfluidic platform with multiplexed electronic detection for spatial tracking of particles
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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.

1. Utformning av Coding elektroder Notera: Figur 1a visar den tre-dimensionella struktur av de micropatterned elektroder. <ol><li> Utforma en uppsättning av fyra 7-bit Gold-koder för kodning av mikroflödessystem kanaler 23. <ol><li> Konstruera två linjära återkopplingsskift-register (LFSR), var och en representerar ett primitivt polynom. </li><li> Använd LFSR att generera en föredragen par 7-bitars m -sequences. </li><li> Cykliskt s...

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Multipla resistiva pulssensorer har tidigare införlivats i mikroflödeschip 28, 29, 30, 31, 32. I dessa system har resistiva pulssensorer antingen inte multiplexeras 28, 29, 30, 31 eller de som krävs för enskilda sensorer att dri...

Noggrann detektering och analys av biologiska partiklar, såsom celler, bakterier eller virus som är suspenderade i vätskan är av stort intresse för en rad tillämpningar 1, 2, 3. Väl matchade i storlek, mikrofluidikanordningar erbjuder unika fördelar för detta ändamål, såsom hög känslighet, mild provmanipulation och välkontrollerad mikro 4, 5, 6, 7. Dessutom kan mikrofluidikanordningar utformas för att använda en kombination av fluiddynamik och kraftfält att passivt fraktionera en heterogen population av biologiska partiklar baserade på olika egenskaper 8, 9, 10, 11, 12. I de anordnings, kan den resulterande partikel användas som utläsning men geografisk information är vanligtvis endast tillgängliga via mikroskopi, vilket begränsar den praktiska nyttan av mikroflödessystem enheten genom att knyta den till ett labb infrastruktur. Därför en integrerad sensor som lätt kan rapportera partiklarnas Spatiotemporal kartläggning, eftersom de är manipulerade på en mikroflödessystem enhet, potentiellt kan aktivera låg kostnad, integrerade lab-on-a-chip enheter som är särskilt attraktiv för testning av prover inom mobil , resursbegränsade inställningar. Tunnfilmselektroder har använts som integrerade sensorer i mikrofluidanordningar för olika tillämpningar 13, 14. Resistiv Pulse Sensing (RPS) är särskilt attraktiv för integrerad avkänning av små partiklar i mikroflödessystem kanaler, eftersom det ger en robust, känslig, och upptäckt mekanism hög genomströmning direkt från elektriska mätningar 15. I RPS, impedansen modulation mellan ett par elektroder, nedsänkt i en elektrolyt, används som ett medel för att detektera en partikel. När partikel passerar genom en öppning, dimensionerad i storleksordningen av partikeln, är antalet och amplituden för övergående pulserna i den elektriska ström som används för att räkna och storlek partiklar, respektive. Vidare kan sensorgeometrin utformas med en fotolitografisk upplösning att forma resistiva pulsvågformer i syfte att öka känsligheten 16, 17, 18, 19 eller för att uppskatta vertikala läget av partiklar i mikrofluidiska kanaler 20. Vi har nyligen infört en skalbar och enkel multiplex resistiv puls sensing teknik som kallas mikroflödes Coded Orthogonal Detection av elektriska Sensing (Mikroflödes koder) 21. Mikroflödes KODER förlitar sig på ensammankopplade nätverk av resistiva pulssensorer, var och en bestående av en matris av elektroder mikrobearbetade för att modulera överledning på ett unikt, urskiljbart sätt, för att möjliggöra multiplexering. Vi har särskilt utformade varje sensor för att producera ortogonala elektriska signaler som liknar de digitala koder som används i koddelad multipelaccess 22 (CDMA) telekommunikationsnät, så att individuella resistiva pulssensorsignalen unikt kan återvinnas från en enda utgångs vågform, även om signaler från olika sensorer stör. På detta sätt, komprimerar vår teknik 2D rumslig information av partiklar in i en 1D elektrisk signal, som medger övervakning av partiklar vid olika platser på ett mikrofluidchip, och samtidigt hålla både enhets- och systemnivå komplexitet till ett minimum. I detta papper presenterar vi ett detaljerat protokoll för experimentella och beräkningsmetoder som krävs för att använda mikroflödes KODER teknik, samt representative resultat från dess användning i analys av simulerade biologiska prover. Med hjälp av resultaten från en prototyp enhet med fyra multiplexerade sensorer som ett exempel för att förklara tekniken ger vi protokoll om (1) den mikro process för att skapa mikroflödes enheter med mikroflödessystem KODER teknik, (2) beskrivning av experimentuppställning inklusive elektroniska, optiska, och fluidisk hårdvara, (3) datoralgoritmen för avkodning interfererande signaler från olika sensorer, och (4) resultaten från detektion och analys av cancerceller i mikrofluidiska kanaler. Vi tror att med hjälp av den detaljerade protokoll som beskrivs här, kan andra forskare använda vår teknik för sin forskning.

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microfluidic platform with multiplexed electronic detection for spatial tracking of particles

Mikroflödes behandling av biologiska prover innebär normalt differential manipulationer av svävande partiklar under olika kraftfält för att rumsligt fraktionera provet bygger på en biologisk egenskap av intresse. För den erhållna rumsliga fördelningen som skall användas som analysavläsningen, är mikrofluidikanordningar ofta utsattes för mikroskopisk analys som kräver komplex instrumentering med högre kostnad och reducerad bärbarhet. Att ta itu med denna begränsning, har vi utvecklat en integrerad elektronisk avkänning teknik för multiplex detektion av partiklar på olika platser på en mikroflödessystem chip. Vår teknik, som kallas Mikroflödes koder kombinerar resistiv Pulse Sensing med Code Division Multiple Access att komprimera 2D rumslig information i en 1D elektrisk signal. I detta papper presenterar vi en praktisk demonstration av mikroflödes KODER teknik för att upptäcka och storlek odlade cancerceller fördelade över flera mikroflödessystem kanaler. Somvaliderats av höghastighets mikroskopi, kan vår teknik noggrant analysera täta cellpopulationer allt elektroniskt utan behov av en extern instrument. Som sådan, kan mikroflödes KODER potentiellt möjliggör låga integrerade lab-on-a-chip enheter som är väl lämpade för point-of-care testning av biologiska prover.

En mikroflödes KODER anordning som består av fyra sensorer fördelade över fyra mikroflödessystem kanaler visas i figur 1b. I detta system, var tvärsnittet av varje mikroflödeskanal utformad för att vara nära till storleken på en cell så att (1) flera celler inte kan passera över elektroderna i parallella och (2) celler förblir nära elektroderna öka känsligheten . Varje sensor är utformad för att generera ett unikt 7-bitars digital kod. Anordningen testa...

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

Vi visar ett mikrofluid plattform med en integrerad ytelektrod nätverk som kombinerar resistiv puls avkänning (RPS) med koddelad multipelaccess (CDMA), för att multiplexera detektering och storleksbestämning av partiklar i multipla mikrofluidikkanaler.

Mikroflödes Plattform med Multiplex elektronisk detektering för rumslig Spårning av partiklar

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

A Microfluidic-based Hydrodynamic Trap for Single Particles

En mikroflödessystem-baserad Hydrodynamisk fälla för enstaka partiklar

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Fluorescence detection methods for microfluidic droplet platforms

Fluorescens detektionsmetoder för mikroflödessystem droppe plattformar

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Bridging the Bio-Electronic Interface with Biofabrication

Att överbrygga Bio-elektroniska gränssnittet mot Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Cell Klämma som en robust, mikroflödes Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Mikroflödes Plattform för Mätning Neutrophil Chemotaxis från Cement helblod

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

En mikroflödessystem plattformen för Precision små volymer Sample Processing och dess användning till storlek Separata biologiska partiklar med en akustisk mikroanordning

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

En mikroflödessystem plattform för hög genomströmning Single-cell isolering och kultur

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

Montering och spårning av mikrobiell gemenskapsutveckling inom en Microwell Array-plattform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

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

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

Imaging rotahåret morfologi av Arabidopsis plantor i en två-lagers mikroflödessystem plattform

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

En mikroflödessystem plattform för längsgående Imaging i Caenorhabditis elegans

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