Fluorescence detection in a micro flow cytometer without on-chip fibers

This paper describes a novel concept of integrated on-chip fiber free laser-induced fluorescence detection system. The poly-dimethylsiloxane (PDMS) chip was fabricated using soft lithography and was bonded with a glass substrate of 150 μm thickness that reduced the distance of channel-to-sidewall to less than 180 μm. The cells and particles detection was conducted by an external single fiber close to the glass substrate that transmitted laser light for simultaneous excitation and receipt of the emission light signals. The performance of the proposed device was demonstrated using fluorescence beads, stained white blood cells, and yeast cells. The experimental results showed the simplicity and flexibility of the proposed device configuration which can provide convenient on-chip integration interface for fast, high throughput, and low-cost laser-induced fluorescence detection micro flow cytometer.     

Integration of thin film amorphous silicon photodetector with lab-on-chip for monitoring protein fluorescence in solution and in live microbial cells

Green fluorescent protein (GFP) and other fluorescent protein variants are widely used as powerful tools to tag and monitor complex biological processes at the cellular, tissue, and organism level. A micrometer-scale GFP detection system is demonstrated in which a p-i-n thin-film amorphous silicon light sensitive layer microfabricated on a glass substrate is integrated with an amorphous silicon carbon alloy absorption filter and an ultra-thin PDMS sheet to detect intracellular expression of GFP. GFP fluorescence was detected in aqueous solution in the nM range, and inside living cells (Escherichia coli) expressing GFP in the 10⁶ cell/mL range. The temporal evolution of the integrated fluorescence from promoter-induced GFP production was monitored in E. coli in solution over 12 h using the detection system. This microfabricated thin-film detection system can potentially be developed into an array-based sensing of GFP allowing for high-throughput and multiplexed analysis of biological and biomedical samples.     

Microfluidic device with dual-channel fluorescence acquisition for quantification/identification of cancer cells

This paper presents an optofluidic device for cell discrimination with two independent interrogation regions. Pumping light is coupled to the device, and cell fluorescence is extracted from the two interrogation zones by using optical fibers embedded in the optofluidic chip. To test the reliability of this device, AU-565 cells—expressing EpCAM and HER2 receptors—and RAMOS cells were mixed in a controlled manner, confined inside a hydrodynamic focused flow in the microfluidic chip and detected individually so that they could be discriminated as positive (signal reception from fluorescently labeled antibodies from the AU-565 cells) or negative events (RAMOS cells). A correlation analysis of the two signals reduces the influence of noise on the overall data.     

A Programmable Biochip for the Applications of Trapping and Adaptive Multisorting Using Dielectrophoresis Array

The adaptive biochip integrating dielectrophoresis (DEP) traps and a programmable multisorting DEP array for the multisorting applications of biomolecules such as proteins and DNA is proposed and demonstrated in this paper. In this research, movable beads are used as the mobile probes to capture the target protein molecules. These beads are chemically modified and immobilized with p50 proteins in our demonstration. An array of micropyramid DEP traps with a good levitation control on the height of the beads is located at the upstream to enhance the hybridization function of the mobile probes. The sample solution mixed with Cy3-I-kappa-B-alpha complex is used in the demonstration. A programmable multisorting DEP array that is located at the downstream sorts out the hybridized beads, which are fluorescently labeled based on the fluorescent detection signals. The magnitude and direction of the DEP force that is applied to the beads with/without labeling fluorescence in the multisorting DEP array are controlled via the distribution of time-variant nonuniform electric fields. The voltage on the individual electrode of the multisorting DEP array is preprogrammed and controlled by a LabVIEW controller with fluorescence detection feedback signals. In contrast to the research of Manaresi et al. [IEEE J. Solid-State Circuits, vol. 38, no. 12, p. 2297, 2003], which was proposed for trapping and sorting beads and cells via Dent traps, to our knowledge, the design of this biochip with the hybridization enhancement via micropyramid DEP traps and the adaptive multisorting DEP array for the mobile probes has never been proposed and implemented to date.     

Portable bead-based fluorescence detection system for multiplex nucleic acid testing: a case study with Bacillus anthracis

This paper describes the design, functioning and use of a portable detection platform for multiplex nucleic acid testing. The system features a bead-supported DNA hybridization assay performed inside a microfluidic cartridge. Polystyrene particles modified with DNA capture probes are confined in the detection area and exposed to a solution of fluorescently labeled target DNA strands. The cartridge, fabricated from inexpensive thermoplastic polymers, allows for conducting up to eight assays in parallel. The detection instrument is equipped with a pneumatic module and a manifold lid serving as an interface to mediate fluid displacement on the cartridge. The fluorescence signal deriving from each assay is recorded by a semi-confocal fluorescence reader embedded in the detection platform. The compact design of the instrument and its level of integration make it possible to obtain an analytical result in less than 15 min, while only few manual steps need to be performed in between. A proof-of-concept demonstration involving Cy3-labeled, PCR-amplified genomic DNA confirms the ability to detect Bacillus anthracis in a multiplexed single-assay format using lef and capC genes. Limits of quantification are on the order of 1 × 10⁹ copies/μL for lef targets.     

Development of a capillary flow microfluidic Escherichia coli biosensor with on-chip reagent delivery using water-soluble nanofibers

Although the potential role of microfluidics in point of care diagnostics is widely acknowledged, the practical limitations to their use still limit deployment. Here, we developed a capillary flow microfluidic with on-chip reagent delivery which combines a lateral flow assay with microfluidic technology. The horseradish peroxidase tagged antibody was electrospun in a water-soluble polyvinylpyrrolidone nanofibers and stored in a microfluidic poly(methyl methacrylate) chip. During the assay, the sample containing Escherichia coli on immunomagnetic beads came in contact with the nanofibers causing them to dissolve and release the reagents for binding. Following hybridization, the solution moved by capillary flow toward a detection zone where the analyte was quantified using chemiluminescence. The limit of detection was found to be approximately 10⁶ CFU/mL of E. coli O157. More importantly, the ability to store sensitive reagents within a microfluidic as nanofibers was demonstrated. The fibers showed almost instant hydration and dissemination within the sample solution.     

A novel integrated microfluidic platform based on micro-magnetic sensor for magnetic bead manipulation and detection

We present a novel integrated microfluidic platform based on micro-magnetic sensor for manipulating and detecting magnetic beads (MB). A micro-spiral planar coil in MB manipulating system microfabricated by micro-electro-mechanical system technology is implemented to manipulate MB, and a giant magnetoimpedance (GMI) based micro-magnetic sensor is employed to detect the trapped MB. In our work, MB can be efficiently trapped by trapping force generated from micro-coil in microchannel. Next, trapped MB are detected by the changing ratio of impedance, as well as the variation of resistance and reactance in GMI sensor for trapped MB induce weak stray magnetic field under the magnetization by external magnetic field. The maximum difference of GMI ratio between with beads condition and without beads condition is 4.0% at the optimum driving frequency of 20 MHz under the external magnetic field of 15 Oe, and resistance ratio varies more significantly than reactance ratio. In comparison with traditional MB detecting methods by GMI sensor, the integrated microfluidic platform based on GMI sensor can not only manipulate and detect MB signal sensitively, but also enhance detection efficiency and decrease the experiment errors. Furthermore, this platform avoids contamination from the solutions in chemically reactive layers and reduces assay time in future biomarker detection. In our work, the microfluidic platform based on GMI sensor has potential applications in biomarker detection via MB manipulation and detection.     

An integrated microfluidic system using mannose-binding lectin for bacteria isolation and biofilm-related gene detection

Molecular diagnosis of biofilm-related genes (BRGs) in common bacteria that cause periprosthetic joint infections may provide crucial information for clinicians. In this study, several BRGs, including ica, fnbA, and fnbB, were rapidly detected (within 1 h) with a new integrated microfluidic system. Mannose-binding lectin (MBL)-coated magnetic beads were used to isolate these bacteria, and on-chip nucleic acid amplification (polymerase chain reaction, PCR) was then performed to detect BRGs. Both eukaryotic and prokaryotic MBLs were able to isolate common bacterial strains, regardless of their antibiotic resistance, and limits of detection were as low as 3 and 9 CFU for methicillin-resistant Staphylococcus aureus and Escherichia coli, respectively, when using a universal 16S rRNA PCR assay for bacterial identification. It is worth noting that the entire process including bacteria isolation by using MBL-coated beads for sample pre-treatment, on-chip PCR, and fluorescent signal detection could be completed on an integrated microfluidic system within 1 h. This is the first time that an integrated microfluidic system capable of detecting BRGs by using MBL as a universal capturing probe was reported. This integrated microfluidic system might therefore prove useful for monitoring profiles of BRGs and give clinicians more clues for their clinical judgments in the near future.     

Methods for counting particles in microfluidic applications

Microfluidic particle counters are important tools in biomedical diagnostic applications such as flow cytometry analysis. Major methods of counting particles in microfluidic devices are reviewed in this paper. The microfluidic resistive pulse sensor advances in sensitivity over the traditional Coulter counter by improving signal amplification and noise reduction techniques. Nanopore-based methods are used for single DNA molecule analysis and the capacitance counter is useful in liquids of low electrical conductivity and in sensing the changes of cell contents. Light-scattering and light-blocking counters are better for detecting larger particles or concentrated particles. Methods of using fluorescence detection have the capability for differentiating particles of similar sizes but different types that are labeled with different fluorescent dyes. The micro particle image velocimetry method has also been used for detecting and analyzing particles in a flow field. The general limitation of microfluidic particle counters is the low throughput which needs to be improved in the future. The integration of two or more existing microfluidic particle counting techniques is required for many practical on-chip applications.     

Characterization and modeling of a microfluidic dielectrophoresis filter for biological species

Microfabricated interdigitated electrode array is a convenient form of electrode geometry for dielectrophoretic trapping of particles and biological entities such as cells and bacteria within microfluidic biochips. We present experimental results and finite element modeling of the holding forces for both positive and negative dielectrophoretic traps on microfabricated interdigitated electrodes within a microfluidic biochip fabricated in silicon with a 12-/spl mu/m-deep chamber. Anodic bonding was used to close the channels with a glass cover. An Experimental protocol was then used to measure the voltages necessary to capture different particles (polystyrene beads, yeast cells, spores and bacteria) against destabilizing fluid flows at a given frequency. The experimental results and those from modeling are found to be in close agreement, validating our ability to model the dielectrophoretic filter for bacteria, spores, yeast cells, and polystyrene beads. This knowledge can be very useful in designing and operating a dielectrophoretic barrier or filter to sort and select particles entering the microfluidic devices for further analysis.     

Autonomous magnetically actuated continuous flow microimmunofluorocytometry assay

This article presents a microfluidic device which integrates autonomous serial immunofluorocytometry binding reactions of cytometric beads with fluorescence detection and quantification in a continuous flow environment. The microdevice assay is intended to alleviate the extensive benchwork and large sample volumes used when conducting traditional immunoassays, without requiring complex external controls. The technology is based on the miniaturization and automation of the serial processing steps of an antigen sandwich immunoassay, with integrated fluorescence detection using paramagnetic microbeads. The continuous flow design may enable temporal tracking of time-varying protein concentrations in a continuously infused sample for clinical applications, specifically for monitoring inflammation marker proteins in blood produced during cardiac surgeries involving cardiopulmonary bypass (CPB) procedures. The device operation was first validated via a single incubation device which measured the concentration of a fluorescently labeled biotin molecule using streptavidin-coated paramagnetic cytometric beads. Subsequently, a dual incubation device was tested with samples of the anaphylatoxin complement protein C3a, and was shown to be capable of differentiating between samples at typical systemic concentrations of the protein (1–5 μg/ml), with very low sample usage (<6 μl/h). It is believed that this continuous flow, automated microimmunosensor technology will be a platform for high sample rate immunoassays capable of tracking and more thoroughly characterizing the systemic inflammation process, and may aid in the development of better treatment options for systemic inflammation during and after CPB.     

Detection of fluorescence from a mixed specimen consisting of micro particles attached to different fluorescent substances using a light waveguide incorporated optical TAS

A “ubiquitous human health care system” will require a monolithic optical total analysis system (TAS) consisting of waveguides and microfluidic channels based on a transparent resin chip. Together with the rapid development of the fluorescent marking method, fluorescence analysis by TAS of mixed-microparticle specimen attached to different fluorescent substances will be necessary. Towards realization of this, we here propose a novel method for using a part of the fluorescence acquired by irradiating microparticles with AC-modulated laser power as light dedicated to the discrimination of fluorescent substances. Since the light power for discrimination was extremely weak, we extracted effective signal components using a lock-in detection method. Then, by comparison with the signal of the original fluorescence, we could determine whether the fluorescence signal was from the microparticles attached to the fluorescent substance to be discriminated. Using a mixed specimen composed of microparticle-attached fluorescent substances with emission peaks of 520 nm and 600 nm, we found that 10% of the acquired fluorescence could successfully determine the specified fluorescent substance as a discrimination signal. The peak value of the discrimination signal was approximately double the amplitude of the stationary noise in the discrimination signal.

     

基于微流控芯片的钙离子浓度检测系统的实现

在细胞内物质定量分析中引入微流控芯片.利用微流控芯片完成细胞的培养、染色、试剂的进给等生物实验功能.设计了用于细胞内钙离子浓度检测的微流控荧光检测系统.通过双波长激发,利用荧光检测系统完成荧光强度和图像的采集.同时研究比值荧光法,计算出定量检测的钙离子浓度.实验结果表明,此检测装置可靠性高,检测结果准确.这一研究,提供了一种细胞检测新手段.为细胞研究提供更加便捷的细胞培养、检测、试剂进给一体化检测装置.     

Simultaneous monitoring of oxygen consumption and acidification rates of a single zebrafish embryo during embryonic development within a microfluidic device

A microfluidic device with a light modulation system was developed to simultaneously measure the oxygen consumption rate (OCR) and acid extrusion rate (AER) of a single zebrafish embryo during embryonic development. The device combines two components: an array of acrylic microwells containing two sensing layers as the dual luminescent sensor for oxygen (O₂) and acid (pH) detection, and a microfluidic module with pneumatically actuated glass lids to controllably seal the microwells. The continuous blue LED and modulated UV LED lights were simultaneously used to excite the dual luminescent sensor, with the emission detected by a single photodetector. The detection signals were then split into DC and AC components to measure the time variations in fluorescence intensity and phosphorescence lifetime for pH and O₂ detection, respectively. We have successfully measured the OCR and AER of a single developing zebrafish embryo inside a sealed microwell from the blastula stage (3 h post-fertilization, 3 hpf) through the hatching stage of 48 hpf. We also demonstrated the measurement of the OCR and AER of a single 48 hpf zebrafish that experienced acute hypoxia by using our device to monitor the transition between aerobic and anaerobic metabolism. We observed that the AER began to significantly increase, while the OCR rapidly decreased after 20 min of hypoxia, indicating the time point of transition where the non-mitochondrial metabolism subsequently dominated the energy production. Our proposed methodology provides the potential for studying the bioenergetic metabolism in a developing organism that relates mitochondrial physiology and disease.     

On-chip PMA labeling of foodborne pathogenic bacteria for viable qPCR and qLAMP detection

Propidium monoazide (PMA) is a membrane impermeable molecule that covalently bonds to double stranded DNA when exposed to light and inhibits the polymerase activity, thus enabling DNA amplification detection protocols that discriminate between viable and non-viable entities. Here, we present a microfluidic device for inexpensive, fast, and simple PMA labeling for viable qPCR and qLAMP assays. The three labeling stages of mixing, incubation, and cross-linking are completed within a microfluidic device that is designed with Tesla structures for passive microfluidic mixing, bubble trappers to improve flow uniformity, and a blue LED to cross-link the molecules. Our results show that the on-chip PMA labeling is equivalent to the standard manual protocols and prevents the replication of DNA from non-viable cells in amplification assays. However, the on-chip process is faster and simpler (30 min of hands-off work), has a reduced likelihood of false negatives, and it is less expensive because it only uses 1/20th of the reagents normally consumed in standard bench protocols. We used our microfluidic device to perform viable qPCR and qLAMP for the detection of S. typhi and E. coli O157. With this device, we are able to specifically detect viable bacteria, with a limit of detection of 7.6 × 10³ and 1.1 × 10³ CFU/mL for S. typhi and E. coli O157, respectively, while eliminating amplification from non-viable cells. Furthermore, we studied the effects of greater flow rates to expedite the labeling process and identified a maximum flow rate of 0.7 μL/min for complete labeling with the current design.     

Driving and sorting of the fluorescent droplets on digital microfluidic platform

In this paper, we present a digital microfluidic droplet sorting platform to achieve automated droplet sorting based on fluorescent detection. We design and fabricate a kind of digital microfluidic chip for manipulating nano-liter-sized liquid droplets, and the chip is integrated with a fluorescence-initiated feedback system for real-time sorting control. The driving and sorting characteristics of fluorescent droplets encapsulating fluorescent-labeled particles are studied on this platform. The droplets dispensed from on-chip reservoir electrode are transported to a fluorescence detection site and sorted according to their fluorescence signals. The fluorescent droplets and non-fluorescent droplets are successfully separated and the number of fluorescent particles inside each droplet is quantified by its fluorescent intensity. We realize droplet sorting at 20 Hz and obtain a linear relationship between the fluorescent particle concentrations and the fluorescence signals. This work is easily adapted for sorting out fluorescent-labeled microparticles, cells and bacteria and thus has the potential of quantifying catalytic or regulatory bio-activities.     

Passive microfluidic devices for plasma extraction from whole human blood

Our goal is to analyze and compare different continuous microfluidic principles dedicated to plasma extraction from hardly diluted human blood for lab-on-chip applications. First, the strengths and weaknesses of various emerging passive microfluidic methods (microfiltration- and centrifugation-based methods) were analyzed. Various devices were designed, microfabricated and tested with beads or blood. Filtration may be efficient, but with a high sample dilution, low flow rate and optimized geometry. Due to fast cell clogging, this remains a short-term solution. Separation effects resulting from centrifugal acceleration in curved channel flows are hindered by Dean vortices and anyhow are not pronounced with blood. An innovative device is then proposed and investigated experimentally. This is based on the lateral migration of red cells and the resulting cell-free layer, which is used to supply geometric singularities (an ear-cavity or a corner-edge) and locally enhance the clear plasma region. A maximum extraction of 10.7% is obtained for 1/20 diluted blood, injected at 100 μL/min in the corner-edge design.     

Microfluidic aptameric affinity sensing of vasopressin for clinical diagnostic and therapeutic applications

We present a microfluidic aptameric biosensor, or aptasensor, for selective detection of clinically relevant analytes with integrated analyte enrichment, isocratic elution and label-free detection by mass spectrometry. Using a microfluidic platform that is coupled to matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), we demonstrate specific purification, enrichment, and label-free detection of trace amounts of arginine vasopressin (AVP), a peptide hormone that is responsible for arterial vasoconstriction. During extreme physical trauma, in particular immunological shock or congestive heart failure, AVP is excreted abnormally and is hence a biomarker for such conditions. The device uses an aptamer, i.e., an oligonucleotide that binds specifically to an analyte via affinity interactions, to achieve highly selective analyte capture and enrichment. In addition, via thermally induced reversible disruption of the aptamer-analyte binding, the device can be easily regenerated for reuse and allows isocratic analyte elution, i.e., release and collection of analytes using a single aqueous solution. Furthermore, the device is coupled to MALDI-MS using a microfluidic flow gate, which directs the eluted analyte onto a MALDI sample plate for mass spectrometry. We first perform systematic characterization of kinetic and thermal release properties, as well as the overall timescale of the assay, using fluorescently labeled AVP. We then demonstrate MALDI-MS detection of unlabeled AVP at clinically relevant concentrations approaching 1 pM.     

Thin-film electrode based droplet detection for microfluidic systems

We report on a droplet-producing microfluidic system with electrical impedance-based detection. The microfluidic devices are made of polydimethylsiloxane (PDMS) and glass with thin film electrodes connected to an impedance-monitoring circuit. Immiscible fluids containing the hydrophobic and hydrophilic phases are injected with syringe pumps and spontaneously break into water-in-oil droplet trains. When a droplet passes between a pair of electrodes in a medium having different electrical conductivity, the resulting impedance change signals the presence of the particle for closed-loop feedback during processing. The circuit produces a digital pulse for input into a computer control system. The droplet detector allows estimation of a droplet's arrival time at the microfluidic chip outlet for dispensing applications. Droplet detection is required in applications that count, sort, and direct microfluidic droplets. Because of their low cost and simplicity, microelectrode-based droplet detection techniques should find applications in digital microfluidics and in three-dimensional printing technology for rapid prototyping and biotechnology.