A structure-free digital microfluidic platform for detection of influenza a virus by using magnetic beads and electromagnetic forces.

H1N1, a subtype of influenza A virus, has emerged as a global threat in the past decades. Due to its highly infectious nature, an accurate and rapid detection assay is urgently required. Therefore, this study presents a new type of digital microfluidic platform for H1N1 virus detection by utilizing a one-aptamer/two-antibodies assay on magnetic beads. The droplets containing magnetic beads were driven by electromagnetic forces on a structure-free, super-hydrophobic surface to automate the entire assay within 40 min. With different levels of hydrophobic modification, the droplets could be easily controlled and positioned without any assisted microstructure. The tunable electromagnetic forces could be adjusted for three kinds of operating modes for the manipulations of beads and droplets, including movement of droplets containing magnetic beads, mixing of two droplets and beads extraction out of droplets. When compared with previous studies, the manipulations of droplets and magnetic particles in this study are more flexible as they can be easily adjusted by fine-tuning the magnetic flux density. Furthermore, the magnetic beads also served as three-dimensional substrates for the new enzyme-linked immunosorbent assay (ELISA)-like assay. The magnetic beads were conjugated with aptamers, which have high specificity towards H1N1 viruses such that they could be specifically captured and detected. The horseradish peroxidase-conjugated secondary antibody was then used to activate tyramide-tetramethylrhodamine (TTMR) such that fluorescent signals could be amplified. With this approach, the limit of detection was experimentally found to be 0.032 hemagglutination units/reaction, which is sensitive enough for clinical diagnostics. This kind of digital microfluidic platform with the ELISA-like assay could effectively reduce the consumption of samples and reagents such that the volume of all droplets including the H1N1 sample, antibodies, TTMR and wash buffers was only 20 µL. This is the first time that a digital microfluidic platform was demonstrated such that the entire diagnostic process for influenza A H1N1 viruses could be performed by using electromagnetic forces, which could be promising for rapid and accurate diagnosis of influenza.

Generating digital drug cocktails via optical manipulation of drug-containing particles and photo-patterning of hydrogels.

An integrated microfluidic system combining 1) an optically-induced-dielectrophoresis (ODEP) module for manipulation of drug-containing particles and 2) an ultraviolet (UV) "direct writing" module capable of patterning hydrogels was established herein for automatic formulation of customized digital drug cocktails. Using the ODEP module, the drug-containing particles were assembled by using moving light patterns generated from a digital projector. The hydrogel, poly(ethylene glycol) diacrylate (PEGDA), was used as the medium in the ODEP module such that the assembled drug-containing particles could be UV-cured and consequently encapsulated in "pills" of specific sizes and shapes by using the UV direct writing module. At an optimal ODEP force of 335 pN, which was achieved in a solution of 15% PEGDA in 0.2 M sucrose, it was possible to manipulate and UV-cure the drug-containing particles. Furthermore, with a digital micromirror device inside the UV direct writing module, different UV patterns could be designed and projected, allowing for the digital drug cocktails to be packaged into different shapes in <60 s. As a demonstration, emulsion droplets containing two different anti-cancer drugs were further tested to show the capability of the developed device. This represents an automatic digital drug cocktail formulating device which stands to revolutionize personalized medicine.

An integrated microfluidic system for rapid detection and multiple subtyping of influenza A viruses by using glycan-coated magnetic beads and RT-PCR.

The influenza A (InfA) virus, which poses a significant global public health threat, is routinely classified into "subtypes" based on viral hemagglutinin (HA) and neuraminidase (NA) antigens. Because there are nearly 200 viral subtypes, current diagnostic approaches require multiplexing or array systems to cover various subtypes of HA and NA. A microfluidic chip featuring a HA × NA array was consequently developed herein for diagnosis and subtyping of InfA viruses via the use of glycan-coated magnetic beads followed by reverse transcription (RT) polymerase chain reaction (PCR). Up to 12 InfA subtypes were simultaneously detected in an automated fashion in less than 100 minutes on this microfluidic platform, representing a significant improvement in analysis speed compared to benchtop RT-PCR and chip-based microarray systems. The limits of detection of the RT-PCR assays ranged from 40 to 3000 copy numbers for the different subtypes of InfA viruses, around two orders of magnitude higher than in previous studies using microfluidic technologies. In summary, the array-type microfluidic chip system provides a rapid, sensitive, and fully automated approach for detection and multiple subtyping of InfA.

Microfluidic platforms for rapid screening of cancer affinity reagents by using tissue samples.

Cancer is the most serious disease worldwide, and ovarian cancer (OvCa) is the second most common type of gynecological cancer. There is consequently an urgent need for early-stage detection of OvCa, which requires affinity reagent biomarkers for OvCa. Systematic evolution of ligands by exponential enrichment (SELEX) and phage display technology are two powerful technologies for identifying affinity reagent biomarkers. However, the benchtop protocols for both screening technologies are relatively lengthy and require well-trained personnel. We therefore developed a novel, integrated microfluidic system capable of automating SELEX and phage display technology. Instead of using cancer cell lines, it is the first work which used tissue slides as screening targets, which possess more complicated and uncovered information for affinity reagents to recognize. This allowed for the identification of aptamer (nucleic acid) and peptide probes specific to OvCa cells and tissues. Furthermore, this developed system could be readily modified to uncover affinity reagents for diagnostics or even target therapy of other cancer cell types in the future.

Detecting miRNA biomarkers from extracellular vesicles for cardiovascular disease with a microfluidic system.

According to World Health Organization reports, cardiovascular diseases (CVDs) are amongst the major causes of death globally and are responsible for over 18 million deaths every year. Traditional detection methods for CVDs include cardiac computerized tomography scans, electrocardiography, and myocardial perfusion imaging scans. Although diagnosis of CVDs through such bio-imaging techniques is common, these methods are relatively costly and cannot detect CVDs in their earliest stages. In contrast, the levels of certain micro RNA (miRNA) biomarkers extracted from extracellular vesicles (EVs) in the bloodstream have been recognized as promising indicators for early CVD detection. However, detection and quantification of miRNA using existing methods are relatively labor-intensive and time-consuming. In this study, a new integrated microfluidic system equipped with highly sensitive field-effect transistors (FETs) was capable of performing EV extraction, EV lysis, target miRNA isolation and miRNA detection within 5 h. The limit of detection was within the physiological range (femtomolar) for two targeted miRNAs, miR-21 and miR-126, meaning that this integrated microfluidic system has the potential to be used as a tool for early detection of CVDs.

A Microfluidic Chip for Detecting Cholangiocarcinoma Cells in Human Bile.

Cholangiocarcinoma (CCA), a biliary tract malignancy, accounts for 20% of all liver cancers. There are several existing methods for diagnosis of CCA, though they are generally expensive, laborious, and suffer from low detection rates. Herein we first developed a means of partially purifying human bile for consequent injection into a microfluidic chip. Then, the novel microfluidic system, which featured 1) a cell capture module, 2) an immunofluorescence (IF) staining module featuring two CCA-specific biomarkers, and 3) an optical detection module for visualization of antibody probes bound to these CCA marker proteins, was used to detect bile duct cancer cells within partially purified bile samples. As a proof of concept, CCA cells were successfully captured and identified from CCA cell cultures, blood samples inoculated with CCA cells, and clinical bile specimens. In 7.5 ml of bile, this system could detect >2, 0, and 1 positive cells in advanced stage patients, healthy patients, and chemotherapy-treated patients, respectively. In conclusion, our microfluidic system could be a promising tool for detection of cancer cells in bile, even at the earliest stages of CCA when cancer cells are at low densities relative to the total population of epithelial cells.

A microfluidic device for antimicrobial susceptibility testing based on a broth dilution method.

Bacterial resistance to antimicrobial compounds is increasing at a faster rate than the development of new antibiotics; this represents a critical challenge for clinicians worldwide. Normally, the minimum inhibitory concentration of an antibiotic, the dosage at which bacterial growth is thwarted, provides an effective quantitative measure for antimicrobial susceptibility testing, and determination of minimum inhibitory concentration is conventionally performed by either a serial broth dilution method or with the commercially available Etest® (Biomerieux, France) kit. However, these techniques are relatively labor-intensive and require a significant amount of training. In order to reduce human error and increase operation simplicity, a simple microfluidic device that can perform antimicrobial susceptibility testing automatically via a broth dilution method to accurately determine the minimum inhibitory concentration was developed herein. As a proof of concept, wild-type (ATCC 29212) and vancomycin-resistant Enterococcus cells were incubated at five different vancomycin concentrations on-chip, and the sample injection, transport, and mixing processes occurred within five reaction chambers and three reagent chambers via the chip's automatic dispensation and dilution functions within nine minutes. The minimum inhibitory concentration values measured after 24h of antibiotic incubation were similar to those calculated using Etest®. With its high flexibility, reliability, and portability, the developed microfluidic device provides a simple method for antimicrobial susceptibility testing in an automated format that could be implemented for clinical and point-of-care applications.

Microfluidics in the selection of affinity reagents for the detection of cancer: paving a way towards future diagnostics.

Microfluidic technologies have miniaturized a variety of biomedical applications, and these chip-based systems have several significant advantages over their large-scale counterparts. Recently, this technology has been used for automating labor-intensive and time-consuming screening processes, whereby affinity reagents, including aptamers, peptides, antibodies, polysaccharides, glycoproteins, and a variety of small molecules, are used to probe for molecular biomarkers. When compared to conventional methods, the microfluidic approaches are faster, more compact, require considerably smaller quantities of samples and reagents, and can be automated. Furthermore, they allow for more precise control of reaction conditions (e.g., pH, temperature, and shearing forces) such that more efficient screening can be performed. A variety of affinity reagents for targeting cancer cells or cancer biomarkers are now available and will likely replace conventional antibodies. In this review article, the selection of affinity reagents for cancer cells or cancer biomarkers on microfluidic platforms is reviewed with the aim of highlighting the utility of such approaches in cancer diagnostics.

An ultrasensitive sandwich type electrochemiluminescence immunosensor for triiodothyronine detection using silver nanoparticle-decorated graphene oxide as a nanocarrier.

An ultrasensitive electrochemiluminescence (ECL) immunosensor was constructed to detect 3,3',5-triiodothyronine (T3). The system employed T3-conjugated, silver nanoparticle-decorated carboxylic graphene oxide (Ag@fGO-T3) as a carrier and anti-T3 antibody-tris(2,2'-bipyridyl) ruthenium(II) (Ru(bpy)3(2+)) as a probe. The Ag@fGO-T3 and Ru(bpy)3(2+) complex could be mobilized rapidly to the anode in the reaction chamber through electrophoresis. The fGO is reduced electrochemically at the electrode, and the electrons could transfer from an anode to the Ru(bpy)3(2+). The complex is excited at the electrode and an ECL signal is produced upon reacting with tripropylamine (TPrA). Because of its large surface area and excellent conductivity, Ag@fGO could enhance ECL signal significantly in the system. Quantitative measurement of T3 could be achieved in the range from 0.1 pg/mL to 0.8 ng/mL with a detection limit of 0.05 pg/mL. In addition, the novel immunosensor showed good specificity in the presence of serum, indicating its high potential in clinical use.

Screening of aptamers specific to colorectal cancer cells and stem cells by utilizing On-chip Cell-SELEX.

Colorectal cancer (CRC) is the most frequently diagnosed cancer around the world, causing about 700,000 deaths every year. It is clear now that a small fraction of CRC, named colorectal cancer stem cells (CSCs) exhibiting self-renewal and extensive proliferative activities, are hard to be eradicated. Unfortunately, highly specific biomarkers for colorectal CSC (CR-CSCs) are lacking that prohibits the development of effective therapeutic strategies. This study designed and manufactured a novel microfluidic system capable of performing a fully automated cell-based, systematic evolution of ligands by exponential enrichment (SELEX) process. Eight CR-CSC/CRC-specific aptamers were successfully selected using the microfluidic chip. Three of the aptamers showed high affinities towards their respective target cells with a dissociation constant of 27.4, 28.5 and 12.3 nM, which are comparable to that of antibodies.

High-efficiency rare cell identification on a high-density self-assembled cell arrangement chip.

Detection of individual target cells among a large amount of blood cells is a major challenge in clinical diagnosis and laboratory protocols. Many researches show that two dimensional cells array technology can be incorporated into routine laboratory procedures for continuously and quantitatively measuring the dynamic behaviours of large number of living cells in parallel, while allowing other manipulations such as staining, rinsing, and even retrieval of targeted cells. In this study, we present a high-density cell self-assembly technology capable of quickly spreading over 300 000 cells to form a dense mono- to triple-layer cell arrangement in 5 min with minimal stacking of cells by the gentle incorporation of gravity and peripheral micro flow. With this self-assembled cell arrangement (SACA) chip technology, common fluorescent microscopy and immunofluorescence can be utilized for detecting and analyzing target cells after immuno-staining. Validated by experiments with real human peripheral blood samples, the SACA chip is suitable for detecting rare cells in blood samples with a ratio lower than 1/100 000. The identified cells can be isolated and further cultured in-situ on a chip for follow-on research and analysis. Furthermore, this technology does not require external mechanical devices, such as pump and valves, which simplifies operation and reduces system complexity and cost. The SACA chip offers a high-efficient, economical, yet simple scheme for identification and analysis of rare cells. Therefore, potentially SACA chip may provide a feasible and economical platform for rare cell detection in the clinic.

A microfluidic chip with a U-shaped microstructure array for multicellular spheroid formation, culturing and analysis.

Multicellular spheroids (MCS), formed by self-assembly of single cells, are commonly used as a three-dimensional cell culture model to bridge the gap between in vitro monolayer culture and in vivo tissues. However, current methods for MCS generation and analysis still suffer drawbacks such as being labor-intensive and of poor controllability, and are not suitable for high-throughput applications. This study demonstrates a novel microfluidic chip to facilitate MCS formation, culturing and analysis. The chip contains an array of U-shaped microstructures fabricated by photopolymerizing the poly(ethylene glycol) diacrylate hydrogel through defining the ultraviolet light exposure pattern with a photomask. The geometry of the U-shaped microstructures allowed trapping cells into the pocket through the actions of fluid flow and the force of gravity. The hydrogel is non-adherent for cells, promoting the formation of MCS. Its permselective property also facilitates exchange of nutrients and waste for MCS, while providing protection of MCS from shearing stress during the medium perfusion. Heterotypic MCS can be formed easily by manipulating the cell trapping steps. Subsequent drug susceptibility analysis and long-term culture could also be achieved within the same chip. This MCS formation and culture platform can be used as a micro-scale bioreactor and applied in many cell biology and drug testing studies.

A simple cell patterning method using magnetic particle-containing photosensitive poly (ethylene glycol) hydrogel blocks: a technical note.

All human organs consist of multiple types of cells organized in a complex pattern to meet specific functional needs. One possible approach for reconstructing human organs in vitro is to generate cell sheets of a specific pattern and later stack them systematically by layer into a three-dimensional organoid. However, many commonly used cell patterning techniques suffer drawbacks such as dependence on sophisticated instruments and manipulation of cells under suboptimal growth conditions. Here, we describe a simple cell patterning method that may overcome these problems. This method is based on magnetic force and photoresponsive poly (ethylene glycol) diacrylate (PEG-DA) hydrogels. The PEG-DA hydrogel was magnetized by mixing with iron ferrous microparticles and then fabricated into blocks with a specific pattern by photolithography. The resolution of the hydrogel empty space pattern was approximately 150 µm and the generated hydrogel blocks can be remotely manipulated with a magnet. The magnetic PEG-DA blocks were used as a stencil to define the area for cell adhesion in the cell culture dish, and the second types of cells could be seeded after the magnetic block was removed to create heterotypic cell patterns. Cell viability assay has demonstrated that magnetic PEG-DA and the patterning process produced negligible effects on cell growth. Together, our results indicate that this magnetic hydrogel-based cell patterning method is simple to perform and is a useful tool for tissue surrogate assembly for disease mechanism study and drug screening.

Down-regulation of UDP-glucose dehydrogenase affects glycosaminoglycans synthesis and motility in HCT-8 colorectal carcinoma cells.

UDP-glucose dehydrogenase (UGDH) catalyzes oxidation of UDP-glucose to yield UDP-glucuronic acid, a precursor of hyaluronic acid (HA) and other glycosaminoglycans (GAGs) in extracellular matrix. Although association of extracellular matrix with cell proliferation and migration has been well documented, the importance of UGDH in these behaviors is not clear. Using UGDH-specific small interference RNA to treat HCT-8 colorectal carcinoma cells, a decrease in both mRNA and protein levels of UGDH, as well as the cellular UDP-glucuronic acid and GAG production was observed. Treatment of HCT-8 cells with either UGDH-specific siRNA or HA synthesis inhibitor 4-methylumbelliferone effectively delayed cell aggregation into multicellular spheroids and impaired cell motility in both three-dimensional collagen gel and transwell migration assays. The reduction in cell aggregation and migration rates could be restored by addition of exogenous HA. These results indicate that UGDH can regulate cell motility through the production of GAG. The enzyme may be a potential target for therapeutic intervention of colorectal cancers.

GC-MS quantification of ketamine, norketamine, and dehydronorketamine in urine specimens and comparative study using ELISA as the preliminary test methodology.

An automated solid-phase extraction procedure combined with the gas chromatography-mass spectrometry (GC-MS) methodology, without derivatization, has been developed for the determination of ketamine (K), norketamine (NK), and dehydronorketamine (DHNK) in urine. The analytical approach is simple and rapid, yet reliable, achieving good linearity (r(2)>0.999 over the concentration range of 30 to 1000 ng/mL), sensitivity (limits of quantification = 15, 10, and 20 ng/mL for K, NK, and DHNK, respectively), accuracy (90-104%), and precision (RSD<8.1%) for all analytes. Two hundred and six urine specimens collected from suspected drug users were analyzed by this protocol and also screened by Neogen ELISA method to evaluate the efficiency as well as the compatibility of these two methods. Neogen ELISA showed high efficiency (98.1%), high sensitivity (90.9%), high specificity (98.9%), low false-positive rate (1.1%), and moderate false-negative rate (9.1%), adopting 10 ng/mL K as the cutoff. Neogen ELISA screening followed by GC-MS analysis appeared to be a good screening-confirmation test scheme for the analysis of K in urine. Twenty of the 22 positive urine specimens contained all three analytes simultaneously, with DHNK showing the highest and K the lowest concentrations.