Microfluidic array cytometer based on refractive optical tweezers for parallel trapping, imaging and sorting of individual cells

Analysis of genetic and functional variability in populations of living cells requires experimental techniques capable of monitoring cellular processes such as cell signaling of many single cells in parallel while offering the possibility to sort interesting cell phenotypes for further investigations. Although flow cytometry is able to sequentially probe and sort thousands of cells per second, dynamic processes cannot be experimentally accessed on single cells due to the sub-second sampling time. Cellular dynamics can be measured by image cytometry of surface-immobilized cells, however, cell sorting is complicated under these conditions due to cell attachment. We here developed a cytometric tool based on refractive multiple optical tweezers combined with microfluidics and optical microscopy. We demonstrate contact-free immobilization of more than 200 yeast cells into a high-density array of optical traps in a microfluidic chip. The cell array could be moved to specific locations of the chip enabling us to expose in a controlled manner the cells to reagents and to analyze the responses of individual cells in a highly parallel format using fluorescence microscopy. We further established a method to sort single cells within the microfluidic device using an additional steerable optical trap. Ratiometric fluorescence imaging of intracellular pH of trapped yeast cells allowed us on the one hand to measure the effect of the trapping laser on the cells' viability and on the other hand to probe the dynamic response of the cells upon glucose sensing.     

Microfluidic liquid-liquid extraction system based on stopped-flow technique and liquid core waveguide capillary

In this work, a miniaturized liquid-liquid extraction system under stopped-flow manipulation mode with spectrometric detection was developed. A Teflon AF liquid-core waveguide (LCW) capillary was used to serve as both extraction channel for organic solvent flow and adsorption detection flow cell. Gravity induced hydrostatic pressure was used to drive the organic and aqueous phases through the extraction channels. During extraction process, a stable organic and aqueous phase interface was formed at the outlet of the capillary, through which the analyte in the flowing aqueous stream was extracted into the stationary organic solvent in capillary. The absorbance of the analyte extracted into the organic solvent was measured in situ by a spectrometric detection system with light emitting diode (LED) as light source and photodiode as absorbance detector. The performance of the system was demonstrated in the determination of sodium dodecyl sulfate (SDS) extracted as an ion pair with methylene blue into chloroform. The precision of the measured absorbance for a 5 mg L⁻¹ SDS standard was 6.1% R.S.D. (n = 5). A linear response range of 1-10 mg L⁻¹ SDS was obtained with 5 min extraction period. The limit of detection (LOD) for SDS based on three times standard deviation of the blank response was 0.25 mg L⁻¹.     

Microfluidic sorting of arbitrary cells with dynamic optical tweezers

Optical gradient forces generated by fast steerable optical tweezers are highly effective for sorting small populations of cells in a lab-on-a-chip environment. The presented system can sort a broad range of different biological specimens by an automated optimisation of the tweezer path and velocity profile. The optimal grab positions for subsequent trap and cell displacements are estimated from the intensity of the bright field image, which is derived theoretically and proven experimentally. We exhibit rapid displacements of 2 μm small mitochondria, yeast cells, rod-shaped bacteria and 30 μm large protoplasts. Reliable sorting of yeast cells in a microfluidic chamber by both morphological criteria and by fluorescence emission is demonstrated.     

Surface transport and stable trapping of particles and cells by an optical waveguide loop

Waveguide trapping has emerged as a useful technique for parallel and planar transport of particles and biological cells and can be integrated with lab-on-a-chip applications. However, particles trapped on waveguides are continuously propelled forward along the surface of the waveguide. This limits the practical usability of the waveguide trapping technique with other functions (e.g. analysis, imaging) that require particles to be stationary during diagnosis. In this paper, an optical waveguide loop with an intentional gap at the centre is proposed to hold propelled particles and cells. The waveguide acts as a conveyor belt to transport and deliver the particles/cells towards the gap. At the gap, the diverging light fields hold the particles at a fixed position. The proposed waveguide design is numerically studied and experimentally implemented. The optical forces on the particle at the gap are calculated using the finite element method. Experimentally, the method is used to transport and trap micro-particles and red blood cells at the gap with varying separations. The waveguides are only 180 nm thick and thus could be integrated with other functions on the chip, e.g. microfluidics or optical detection, to make an on-chip system for single cell analysis and to study the interaction between cells.     

Microfluidic flow rate detection based on integrated optical fiber cantilever

We demonstrate an integrated microfluidic flow sensor with ultra-wide dynamic range, suitable for high throughput applications such as flow cytometry and particle sorting/counting. A fiber-tip cantilever transduces flow rates to optical signal readout, and we demonstrate a dynamic range from 0 to 1500 microL min(-1) for operation in water. Fiber-optic sensor alignment is guided by preformed microfluidic channels, and the dynamic range can be adjusted in a one-step chemical etch. An overall non-linear response is attributed to the far-field angular distribution of single-mode fiber output.     

Light emitting diode based flow-through optical absorption detectors

Simple inexpensive high performance optical absorption detectors are possible using light emitting diodes (LEDs) as light sources. The designs presented in the literature are reviewed. Designs used by the investigators are described in detail with respect to construction, electronic design, performance and cost; these have not previously been described in the literature. Characteristics of commercially available LEDs are tabulated. At the simple end, a single beam, dc driven, transmittance output detector can be constructed within the body of a LED. At the high performance end, fully referenced, computer interfaced detectors are described that are pulsed at high speeds to attain measurement standard deviations in the range of 2-3 x 10(-6) absorbance.     

Microfluidic sorting of fluorescently activated cells depending on gene expression level

During the last few years, fluorescence activated cell sorter has played an important role in a variety of biological investigations as well as clinical diagnostics. However, the conventional fluorescence activated cell sorter has several limitations, such as large size, large sample volumes required for operation, and high cost. In this paper, we present a novel microfluidic device that can separate cells based on various fluorescent protein expression levels. Our system consists of three major parts: focusing, detection, and separation. The operating principles are briefly as follows: first fluorescent cells were delivered into the microfluidic chip and focused in the center of channel by sheath flow. Subsequently, the cells were excited by a 532 nm laser at 30 μW and concurrently detected by a photomultiplier tube. Based on their fluorescence intensities, the cells were separated into three outlets by a dielectrophoretic force. Using this system, we successfully separated the genetically modified cells at 0.1 μL/min (sample flow rate) to sheath flow rate at 1:5, 5 Vpp voltage, and 800 kHz frequency. The separation efficiency was measured as high as 94.7%. In conclusion, we found that our system has the capability of separating genetically modified cells with various fluorescent intensities and help study biology and medicine in a molecular level.     

Amplified spontaneous emission,optical waveguide and polarized emission based on 2,5-diaminoterephthalates

A series of 2,5-diaminoterephthalates with a simple structure were synthesized through one-step reaction, and their bar-shaped single crystals with a large size and a smooth surface have been obtained via the solvent-evaporation method. These crystals exhibit bright emission with fluorescence quantum yields higher than 0.2. They display the waveguide property, and low optical loss coefficients for waveguide have been determined for the crystal of one compound. In addition, the crystal can cause linear polarization of the light emitted from it, with a high polarization contrast of 0.70. Most importantly, these crystals can realize amplified spontaneous emission(ASE), including the red ASE, with appreciable energy thresholds of 72–198 k W/cm~2 and high gain coefficients, which suggests the potential of these crystals for the application in organic solid-state lasers.     

Microfluidic system for dielectrophoretic separation based on a trapezoidal electrode array

This paper presents a novel microfluidic device for dielectrophoretic separation based on a trapezoidal electrode array (TEA). In this method, particles with different dielectric properties are separated by the device composed of the TEA for the dielectrophoretic deflection of particles under negative dielectrophoresis (DEP) and poly(dimethylsiloxane)(PDMS) microfluidic channel with a sinuous and expanded region. Polystyrene microparticles are exposed to an electric field generated from the TEA in the microfluidic channel and are dielectrophoretically focused to make all of them line up to one sidewall. When these particles arrive at the region of another TEA for dielectrophoretic separation, they are separated having different positions along the perpendicular direction to the fluid flow due to their different dielectrophoretic velocities. To evaluate the separation process and performance, both the effect of the flow rate on dielectrophoretic focusing and the influence of the number of trapezoidal electrodes on dielectrophoretic separation are investigated. Now that this method utilizes the TEA as a source of negative DEP, non-specific particle adhering to the electrode surface can be prevented; conventional separation approaches depending on the positive DEP force suffer from this problem. In addition, since various particle types are continuously separated, this method can be easily applicable to the separation and analysis of various dielectric particles with high particle recovery and selectivity.     

Microfluidic chip-based liquid-liquid extraction and preconcentration using a subnanoliter-droplet trapping technique

A robust and simple approach for microfluidic liquid-liquid (L-L) extraction at the subnanoliter-scale was developed for on-chip sample pretreatment. Organic solvent droplets of a few hundred pL were trapped within micro recesses fabricated in the channel walls of a microfabricated glass chip. L-L extraction was performed by delivering aqueous samples through the channel, with the sample stream continuously flowing adjacent to the droplets. The analytes in aqueous streams were enriched within the droplet with high preconcentration factors owing to both phase transfer and dissolution of organic solvent into the bypassing aqueous sample. An aqueous solution of butyl rhodamine B (BRB) and 1-hexanol were used, respectively, as sample and extractant to demonstrate the performance of the system. The fluorescence intensity of the dye extracted into the droplet was monitored in situ by LIF. The system proved to be an efficient means for achieving high enrichment factors of over 1000, with sample consumption of a few microL. Quantitative measurement of the extracted analyte was achieved with a linear response in the range 1 x 10(-9)-8 x 10(-7) M BRB. The precision of the measured fluorescence values for a 10(-7) M BRB standard with a 12.5 min preconcentration period was 6.6% RSD (n = 5).     

Natural gas pipeline leak detector based on NIR diode laser absorption spectroscopy

The paper reports on the development of an integrated natural gas pipeline leak detector based on diode laser absorption spectroscopy. The detector transmits a 1.653 microm DFB diode laser with 10 mW and detects a fraction of the backscatter reflected from the topographic targets. To eliminate the effect of topographic scatter targets, a ratio detection technique was used. Wavelength modulation and harmonic detection were used to improve the detection sensitivity. The experimental detection limit is 50 ppmm, remote detection for a distance up to 20 m away topographic scatter target is demonstrated. Using a known simulative leak pipe, minimum detectable pipe leak flux is less than 10 ml/min.     

Effects of optical trapping and liquid surface deformation on the laser microdeposition of a polymer assembly in solution

A polymer microassembly is formed by focusing a near-infrared (NIR) laser beam in a thin film of a polymer solution. We have investigated the mechanism of laser microdeposition of a polyfluorene assembly by measuring the surface deformation of the solution film and the morphology of the deposited assembly. It is clearly observed that a rupture is formed at the laser focus in the solution film by using laser interferometric imaging. The time necessary for the rupture formation and the volume of the deposited microassembly are analyzed as a function of laser power. Experimental results suggest that the solution surface deformation induced by local laser heating and optical trapping effects determined the volume of the laser microdeposition. By combining this method with multiple optical trapping, a polymer microassembly with a polygonal morphology is formed on the glass substrate.     

Analysis of single-cell differences by use of an on-chip microculture system and optical trapping

A method is described for continuous observation of isolated single cells that enables genetically identical cells to be compared; it uses an on-chip microculture system and optical tweezers. Photolithography is used to construct microchambers with 5-microm-high walls made of thick photoresist (SU-8) on the surface of a glass slide. These microchambers are connected by a channel through which cells are transported, by means of optical tweezers, from a cultivation microchamber to an analysis microchamber, or from the analysis microchamber to a waste microchamber. The microchambers are covered with a semi-permeable membrane to separate them from nutrient medium circulating through a "cover chamber" above. Differential analysis of isolated direct descendants of single cells showed that this system could be used to compare genetically identical cells under contamination-free conditions. It should thus help in the clarification of heterogeneous phenomena, for example unequal cell division and cell differentiation.     

Monitoring cell survival after extraction of a single subcellular organelle using optical trapping and pulsed-nitrogen laser ablation

This paper characterizes cell viability in three different cell lines--Chinese hamster ovary cells (CHO), neuroblastoma cells fused with glialoma cells (NG108-15) and murine embryonic stem cells (ES-D3)--after N2 laser disruption of the cell membrane and removal, via optical trapping, of a single subcellular organelle. Morphological changes and viability (as determined by live/dead fluorescent stains) of the cell were monitored every half hour over a 4-h period postsurgery. The ability of the cell to survive organelle extraction was found to depend both on the conditions under which surgery was performed and on the cell type. The average viability after surgery for CHO cells was approximately 80%, for NG 108 cells it was approximately 30% and for ES-D3 cells postsurgery viability was approximately 10%. From over 600 surgeries we found the survival of the cell is determined almost exclusively within the first hour postsurgery regardless of cell line. The optimal pulse energy for N2 laser ablation was approximately 0.7 microJ. The N2 pulse produced an approximately 1-3 microm hole in the cell membrane and proved to be the primary source of cell death in those cells that did not survive the procedure.     

Characterization of an element selective GC-plasma detector based on diode laser atomic absorption spectrometry

An element-selective GC-plasma detector consisting of a modulated low-pressure microwave-induced plasma and a laser detection system based on Wavelength Modulation Diode Laser Atomic Absorption Spectroscopy (WM-DLAAS) was investigated. The influence on the plasma conditions and the fragmentation capability of different types of organic compounds, such as alkanes, alkenes, alkynes and aromatics, was studied by measurements of the element ratios of C, H and Cl, and the population density of the metastable 1s₅ level of Kr added as a trace to the plasma gas (He or Ar). Deviations from the expected stoichiometrical ratios were found to be insignificant. Therefore, calibration and determination of the sum formula of analyte species is possible if an internal standard is used. Furthermore, the correlation between the metastable density in the microwave-induced plasma and the total dissociation energy of the analytes was studied.     

Microfluidic large-scale integration on a chip for mass production of monodisperse droplets and particles

In this study, we report the mass production of monodisperse emulsion droplets and particles using microfluidic large-scale integration on a chip. The production module comprises a glass microfluidic chip with planar microfabricated 16-256 droplet-formation units (DFUs) and a palm-sized stainless steel holder having several layers for supplying liquids into the inlets of the mounted chip. By using a module having 128 cross-junctions (i.e., 256 DFUs) arranged circularly on a 4 cm x 4 cm chip, we could produce droplets of photopolymerizable acrylate monomer at a throughput of 320.0 mL h(-1). The product was monodisperse, having a mean diameter of 96.4 microm, with a coefficient of variation (CV) of 1.3%. Subsequent UV polymerization off the module yielded monodisperse acrylic microspheres at a throughput of approximately 0.3 kg h(-1). Another module having 128 co-flow geometries could produce biphasic Janus droplets of black and white segments at 128.0 mL h(-1). The product had a mean diameter of 142.3 microm, with a CV of 3.3%. This co-flow module could also be applied in the mass production of homogeneous monomer droplets.     

Slow motion, trapping, and sorting of water- and chloroform-soluble porphyrins in nanowells

A two-step self-assembly procedure on smooth, aminated silica particles established holey monolayers. At first, single, flat-lying porphyrin tetraamides (A) were bound covalently, followed by the build-up of a rigid monolayer made of diamido bolaamphiphiles (bolas) around the porphyrin islands. "Nanowells" around porphyrin (A) bottoms with a uniform diameter of 2.2 nm and varying depths of 0.6, 1.0, or 1.5 nm depending on the length of the applied bolas were thus obtained. Oligoethylene headgroups solubilized the particles in water, ethanol, and chloroform/ethanol, and two hydrogen bond chains between the secondary amide groups prevented swelling of the monolayer. Manganese(III) porphyrinates (B) migrated from the bulk solution to the bottom of the form-stable nanowells with a speed of about 1 pm/s and were trapped there above porphyrin (A). After isolation of the (A,B) particles by centrifugation or ultrafiltration, the particles were suspended in a chloroform solution of a chlorin (C), which was also fixated irreversibly on the bottom of the nanowells. The nanowells thus contained three different porphyrins A,B,C in a noncovalent stack. The reverse sequence A,C,B was built-up correspondingly, first in chloroform/ethanol, and then in water. The "sorting" of A,B,C and A,C,B systems was characterized by visible spectra, sequence-dependent fluorescence quenching, and cyclic voltammetry of the top component. The molecular sorting method is the first of its kind and should be generally useful for the production of noncovalent reaction systems on any smooth surface.     

Biosensor for L-phenylalanine based on the optical detection of NADH using a UV light emitting diode

A biosensor was developed for the detection of L-phenylalanine (Phe) and demonstrated for use in the diagnosis of phenylketonuria (PKU). It consists of L-phenylalanine dehydrogenase (L-PheDH) immobilized on a membrane, an ultraviolet light-emitting diode excitation system, and a photomultiplier tube. The L-PheDH was immobilized on a teflon membrane modified with 2-methacryloyloxyethyl phosphorylcholine and placed at the distal and of an optical fiber. The concentration of Phe was determined by immersing the sensor into a sample solution that also contained NAD⁺ and measurement of the fluorescence of the NADH produced by enzymatic reaction. Two L-PheDHs (from Thermoactinomyces intermedius and Sporosarcina sp.) were studied and compared. The fluorescence intensities of the biosensor are linearly related to the L-Phe concentrations in the range from 10 μmol L^?1 to 10 mmol L^?1. The sensor also was operated in the kinetic mode by differential determination of the slope of the signal within 2 min. The analytical range of the sensor is adequate for application in the genotypic diagnosis of PKU (diagnostic value >600 μmol L^?1). High sensitivity, good cost-benefit ratio, and low power consumption are typical features of this biosensing system that can can be applied to routine screening of newborn.

A low-cost light-emitting diode induced fluorescence detector for capillary electrophoresis based on an orthogonal optical arrangement

In this work, a simple and low-cost miniaturized light-emitting diode induced fluorescence (LED-IF) detector based on an orthogonal optical arrangement for capillary electrophoresis (CE) was developed, using a blue concave light-emitting diode (LED) as excitation source and a photodiode as photodetector. A lens obtained from a waste DVD-ROM was used to focus the LED light beam into an ∼80 μm spot. Fluorescence was collected with an ocular obtained from a pen microscope at 45° angle, and passed through a band-pass filter to a photodiode detector. The performance of the LED-IF detector was demonstrated in CE separations using sodium fluorescein and fluorescein isothiocyanate (FITC)-labeled amino acids as model samples. The limit of detection for sodium fluorescein was 0.92 μM with a signal-to-noise ratio (S/N) of 3. The total cost of the LED-IF detector was less than $ 50.     

Microfluidic integration of substantially round glass capillaries for lateral patch clamping on chip

High-throughput screening of drug candidates for channelopathies can greatly benefit from an automated patch-clamping assay. Automation of the patch clamping through microfluidics ideally requires on-chip integration of glass capillaries with substantially round cross section. Such round capillaries, if they can only be integrated to connect isolated reservoirs on a substrate surface, will lead to a "lateral" configuration which is simple yet powerful for the patch clamping. We demonstrate here "lateral" patch clamping through microfluidic integration of substantially round glass capillaries in a novel process. The process adopts two well-known phenomena from microelectronics: keyhole-void formation and thermal-reflow of phosphosilicate glass in silicon trenches. The process relies on the same physical principle as the preparation of conventional micropipette electrodes by heat-pulling and fire-polishing glass tubes. The optimized process forms capillaries with a diameter approximately 1.5 microm and variation <10%. Functionality of the integrated glass capillaries for the patch-clamp recording has been verified by statistical test results from a sample of one hundred capillaries on mammalian cells (RBL-1) in suspension: 61% formed gigaseals (>1 GOmega) and of those approximately 48% (29% of all) achieved whole-cell recordings. Pharmacological blockade of ion channel activity and longevity of a whole-cell mode on these capillaries have also been presented.