Enhancing protease activity assay in droplet-based microfluidics using a biomolecule concentrator

We introduce an integrated microfluidic device consisting of a biomolecule concentrator and a microdroplet generator, which enhances the limited sensitivity of low-abundance enzyme assays by concentrating biomolecules before encapsulating them into droplet microreactors. We used this platform to detect ultralow levels of matrix metalloproteinases (MMPs) from diluted cellular supernatant and showed that it significantly (~10-fold) reduced the time required to complete the assay and the sample volume used.     

Quantitative detection of protein expression in single cells using droplet microfluidics

We demonstrate that single cells can be controllably compartmentalized within aqueous microdroplets; using such an approach we perform high-throughput screening by detecting the expression of a fluorescent protein in individual cells with simultaneous measurement of droplet size and cell occupancy.     

Electrochemical droplet-based microfluidics using chip-based carbon paste electrodes for high-throughput analysis in pharmaceutical applications

This paper presents the first example of a pharmaceutical application of droplet-based microfluidics coupled with chronoamperometric detection using chip-based carbon paste electrodes (CPEs) for determination of dopamine (DA) and ascorbic acid (AA). Droplets were generated using an oil flow rate of 1.80 μL min⁻¹, whereas a flow rate of 0.80 μL min⁻¹ was applied to the aqueous phase, which resulted in a water fraction of 0.31. The optimum applied potential for chronoamperometric measurements in droplets was found to be 150 mV. Highly reproducible analysis of DA and AA was achieved with relative standard deviations of less than 5% for both intra-day and inter-day measurements. The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 20 and 70 μM for DA and 41 and 137 μM for AA, respectively. Linearity of this method was in the ranges of 0.02–3.0 mM for DA and 0.04–3.0 mM for AA. This system was successfully applied to determine the amounts of DA and AA in intravenous drugs. Calibration curves of DA and AA for quantitative analysis were obtained with good linearity with R² values of 0.9984 and 0.9988, respectively. Compared with the labeled amounts, the measured concentrations of DA and AA obtained from this system were insignificantly different, with error percentages of less than ±3.0%, indicating a high accuracy of the developed method.     

Concentration and binary separation of micro particles for droplet-based digital microfluidics

This paper describes a concept of concentration and binary separation of particles and its experimental confirmations for digital microfluidics where droplets are driven by the mechanism of electrowetting-on-dielectric (EWOD). As a fundamental separation unit, a binary separation scheme is developed, separating two different types of particles in one droplet into two droplets, one type each. The separation scheme consists of three distinctive steps, each with their own challenges: (1) isolate two different types of particles by electrophoresis into two regions inside a mother droplet, (2) physically split the mother droplet into two daughter droplets by EWOD actuation so that each type of particle is concentrated in each daughter droplet, and (3) free the daughter droplets from the separation site by EWOD to ready them for follow-up microfluidic operations. By applying a similar procedure to a droplet containing only one type of particle, two daughter droplets of different particle concentrations can be created. Using negatively charged carboxylate modified latex (CML) particles, 83% of the total particles are concentrated in a daughter droplet. Successful binary separation is also demonstrated using negatively charged CML particles and no-charge-treated polystyrene particles. Despite the undesired vortex developed inside the mother droplet, about 70% of the total CML particles are concentrated in one daughter droplet while about 70% of the total polystyrene particles are concentrated in the other daughter droplet.     

Particles and microfluidics merged: perspectives of highly sensitive diagnostic detection

There is a growing need for diagnostic technologies that provide laboratories with solutions that improve quality, enhance laboratory system productivity, and provide accurate detection of a broad range of infectious diseases and cancers. Recent advances in micro- and nanoscience and engineering, in particular in the areas of particles and microfluidic technologies, have advanced the “lab-on-a-chip” concept towards the development of a new generation of point-of-care diagnostic devices that could significantly enhance test sensitivity and speed. In this review, we will discuss many of the recent advances in microfluidics and particle technologies with an eye towards merging these two technologies for application in medical diagnostics. Although the potential diagnostic applications are virtually unlimited, the most important applications are foreseen in the areas of biomarker research, cancer diagnosis, and detection of infectious microorganisms.

Lab-chip HPLC with integrated droplet-based microfluidics for separation and high frequency compartmentalisation

We demonstrate the integration of a droplet-based microfluidic device with high performance liquid chromatography (HPLC) in a monolithic format. Sequential operations of separation, compartmentalisation and concentration counter were conducted on a monolithic chip. This describes the use of droplet-based microfluidics for the preservation of chromatographic separations, and its potential application as a high frequency fraction collector.     

Immunomagnetic separation and rapid detection of bacteria using bioluminescence and microfluidics

This paper describes an immunomagnetic separation of target bacterial cells from others by using magnetic bead. The surface of bead was coated with antibodies which can capture specific organism. The binding efficiency of immunomagnetic bead (IMB) capturing target bacterial cells was higher than 98% when the concentrations of target and interferent bacterial cells were at the same level. The concentration of bacteria was determined indirectly by detecting adenosine 5′-triphosphate (ATP) employing bioluminescence (BL) reaction of firefly luciferin-ATP. Benzalkonium chloride (BAC) was used as an ATP extractant from living bacterial cells. We found that BAC could enhance the light emission when the concentration of BAC was less than 5.3 × 10⁻²% (w/v) and the BL intensity reached its maximum at the concentration of BAC was 2.7 × 10⁻²%, which was 10-fold stronger than that without BAC. Based on the principle of the IMB, a microfluidic chip combined with immunofluorescence assay for separating and detecting bacteria simultaneously was also developed. The IMBs were magnetically fixed in the bead-beds of chip channels with a 3-mm diameter of NdFeB permanent magnet. The target bacterial cells can be captured magnetically and observed by a fluorescent microscope.     

Highly sensitive rare cell detection based on quantum dot probe fluorescence analysis

This study presents an efficient and sensitive method for detecting rare cells without cell culture, in which cells are analyzed quantitatively using quantum dots (QDs) as a fluorescent probe. By the conjugation of QDs with cells, the biotin–streptavidin reaction functions as a bridge to connect QDs and cells. The cells can be quantified based on the correlation of the QD fluorescence intensity with the cell population. Non-specific adsorption and cross-reaction of QD625–streptavidin on T cell membrane are neglected by reacting with biotin anti-human CD3 and mixing with red blood cell, respectively. Additionally, the photo-activation period and pH can be controlled to enhance the fluorescence of cell populations, which increases linearly with the number of T cells from 40 to 100,000, not only in a single T cell line but also in mixing with a total of 10⁶ red blood cells. Moreover, the specific T cells can be detected in less than 15 min, even though rare specific cells may number only 40 cells. Among the advantages, the proposed system for detecting rare cells include simplicity of preparation, low cost, rapid detection, and high sensitivity, all of which can facilitate the detection of circulating tumor cells in early stages of diagnosis or prognosis.      

Quantitative detection of tetracycline residues in honey by a simple sensitive immunoassay

Tetracyclines (TCs) are widely used for prevention and control of infectious diseases and have a great activity against variety of Gram-positive and Gram-negative bacteria. Due to the widespread use of TCs in animal husbandry, it can lead to an increase the risk of TCs remaining in human food. To protect consumers, many countries have set acceptable tolerance levels for these drugs. Therefore, it is necessary to establish a suitable analytical technique with specificity, sensitivity and simplicity.The biotin-avidin mediated ELISA method was performed to determine TC residues in honey quantitatively. By using PBS-EDTA assay buffer at pH 7.2, a honey solution of TC standard was prepared and diluted. And no additional pre-treatment of sample was required in this method. The limit of detection and limit of quantitation of the optimized method were 3.98 × 10⁻¹⁰ M (0.19 μg L⁻¹) and 7.94 × 10⁻¹⁰ M (0.38 μg L⁻¹), respectively, and the dynamic range was from 1.52 μg L⁻¹ to 152 μg L⁻¹ of TC in honey. No cross-reactivity was observed with the structurally similar compounds, and mean percent recoveries of TC spiked in honey ranged from 95% to 101%. Compared to other methods, this method was superior in terms of detection limit, dynamic range, and % recovery with simple sample-preparation.     

Contactless conductivity detection for microfluidics: designs and applications

Different methods for construction of contactless conductivity detectors (CCD) for microchip electrophoresis device are described in this review. This includes three main schemes of CCD for microchips, such as (i) the detection electrodes are placed along the microchannel from outside of the microchip and they are insulated from the channel by the cover lid of microchip device; (ii) the electrodes are placed across of the microchannel in the same plane and they are insulated by thin separation channel walls and (iii) electrodes are buried in widened part of microchannel and they are insulated from solution by ultrathin layer of silicon carbide. Specific issues related to the CCD on microfluidics are discussed, such as an influence of shape and magnitude of ac voltage and placement of electrodes and their insulation. Various applications for security, pharmacological, bioassays and food analysis purposes are described.     

Highly sensitive and selective electrochemical detection of L-cysteine using nanoporous gold

Nanoporous gold (NPG) with uniform pore sizes and ligaments was prepared by using a simple dealloying method. NPG electrodes exhibit excellent electrocatalytic activity towards the oxidation of CySH and the mechanism for the electrochemical reaction of CySH on NPG has been discussed. Interestingly, if the operating potential is fixed at 0.65 V, a strong current is observed and interferences by tryptophan and tyrosine are avoided. The calibration plot is linear in the concentration range from 1 μM to 400 μM (R²?=?0.994), and the quantification limit is as low as 50 nM. The NPG-modified electrode has good reproducibility, high sensitivity and selectivity, can be used to sense CySH in aqueous solution.

Highly sensitive biomolecular fluorescence detection using nanoscale ZnO platforms

Fluorescence detection is currently one of the most widely used methods in the areas of basic biological research, biotechnology, cellular imaging, medical testing, and drug discovery. Using model protein and nucleic acid systems, we demonstrate that engineered nanoscale zinc oxide structures can significantly enhance the detection capability of biomolecular fluorescence. Without any chemical or biological amplification processes, nanoscale zinc oxide platforms enabled increased fluorescence detection of these biomolecules when compared to other commonly used substrates such as glass, quartz, polymer, and silicon. The use of zinc oxide nanorods as fluorescence enhancing substrates in our biomolecular detection permitted sub-picomolar and attomolar detection sensitivity of proteins and DNA, respectively, when using a conventional fluorescence microscope. This ultrasensitive detection was due to the presence of ZnO nanomaterials which contributed greatly to the increased signal-to-noise ratio of biomolecular fluorescence. We also demonstrate the easy integration potential of zinc oxide nanorods into periodically patterned nanoplatforms which, in turn, will promote the assembly and fabrication of these materials into multiplexed, high-throughput, optical sensor arrays. These zinc oxide nanoplatforms will be extremely beneficial in accomplishing highly sensitive and specific detection of biological samples involving nucleic acids, proteins and cells, particularly under detection environments involving extremely small sample volumes of ultratrace-level concentrations.     

Microspot-based ELISA in microfluidics: chemiluminescence and colorimetry detection using integrated thin-film hydrogenated amorphous silicon photodiodes

Microfluidic technology has the potential to decrease the time of analysis and the quantity of sample and reactants required in immunoassays, together with the potential of achieving high sensitivity, multiplexing, and portability. A lab-on-a-chip system was developed and optimized using optical and fluorescence microscopy. Primary antibodies are adsorbed onto the walls of a PDMS-based microchannel via microspotting. This probe antibody is then recognised using secondary FITC or HRP labelled antibodies responsible for providing fluorescence or chemiluminescent and colorimetric signals, respectively. The system incorporated a micron-sized thin-film hydrogenated amorphous silicon photodiode microfabricated on a glass substrate. The primary antibody spots in the PDMS-based microfluidic were precisely aligned with the photodiodes for the direct detection of the antibody-antigen molecular recognition reactions using chemiluminescence and colorimetry. The immunoassay takes ~30 min from assay to the integrated detection. The conditions for probe antibody microspotting and for the flow-through ELISA analysis in the microfluidic format with integrated detection were defined using antibody solutions with concentrations in the nM-μM range. Sequential colorimetric or chemiluminescence detection of specific antibody-antigen molecular recognition was quantitatively detected using the photodiode. Primary antibody surface densities down to 0.182 pmol cm(-2) were detected. Multiplex detection using different microspotted primary antibodies was demonstrated.     

Rapid and sensitive DNA target detection using enzyme amplified electrochemical detection based on microchip

A rapid and sensitive DNA targets detection using enzyme amplified electrochemical detection (ED) based on microchip was described. We employed a biotin‐modified DNA, which reacted with avidin‐conjugated horseradish peroxidase (avidin–HRP) to obtain the HRP‐labeled DNA probe and hybridized with its complementary target. After hybridization, the mixture containing dsDNA‐HRP, excess ssDNA‐HRP, and remaining avidin–HRP was separated by MCE. The separations were performed at a separation voltage of +1.6 kV and were completed in less than 100 s. The HRP was used as catalytic labels to catalyze H₂O₂/o‐aminophenol reaction. Target DNA could be detected by the HRP‐catalyzed reduction with ED. With this protocol, the limits of quantification for the hybridization assay of 21‐ and 39‐mer DNA fragments were of 8×10^?12 M and 1.2×10^?11 M, respectively. The proposed method has been applied satisfactorily in the analysis of Escherichia coli genomic DNA. We selected the detection of PCR amplifications from the gene of E. coli to test the real applicability of our method. By using an asymmetric PCR protocol, we obtained ssDNA targets of 148 bp that could be directly hybridized by the single‐stranded probe and detected with ED.     

Controlled synthesis of nonspherical microparticles using microfluidics

The controlled synthesis of nonspherical microparticles using microfluidics processing is described. Polymer droplets, formed by shearing a photopolymer using a continuous water phase at a T-junction, were constrained to adopt nonspherical shapes by confining them using appropriate microchannel geometries. Plugs were obtained by shearing the polymer phase at low shear rates, while disks were obtained by flattening droplets using a channel of low height. The nonspherical shapes formed were permanently preserved by photopolymerizing the constrained droplets in situ using ultraviolet light. Monodisperse plugs and disks of different lengths and diameters were obtained by varying the flow rates of the two phases.     

Highly sensitive smartphone-based detection of Listeria monocytogenes using SYTO9

Listeriosis is caused by Listeria monocytogenes(LM) and is currently considered to be one of the leading food-borne diseases worldwide, with mortality rate of 20%～30%. Currently, detection methods for LM are time-consuming with low sensitivity, and delayed detection results. SYTO9 has a high affinity for DNA and exhibits enhanced fluorescence upon binding. Therefore, this study used SYTO9 staining and image processing to develop a rapid loop mediated isothermal amplification(LAMP) detection meth...