Nanomanipulation of single influenza virus using dielectrophoretic concentration and optical tweezers for single virus infection to a specific cell on a microfluidic chip

A major problem when analyzing bionanoparticles such as influenza viruses (approximately 100 nm in size) is the low sample concentrations. We developed a method for manipulating a single virus that employs optical tweezers in conjunction with dielectrophoretic (DEP) concentration of viruses on a microfluidic chip. A polydimethylsiloxane microfluidic chip can be used to stably manipulate a virus. The chip has separate sample and analysis chambers to enable quantitative analysis of the virus functions before and after it has infected a target cell. The DEP force in the sample chamber concentrates the virus and prevents it from adhering to the glass substrate. The concentrated virus is transported to the sample selection section where it is trapped by optical tweezers. The trapped virus is transported to the analysis chamber and it is brought into contact with the target cell to infect it. This paper describes the DEP virus concentration for single virus infection of a specific cell. We concentrated the influenza virus using the DEP force, transported a single virus, and made it contact a specific H292 cell.     

A paper-based microfluidic Dot-ELISA system with smartphone for the detection of influenza A

An automated, portable, and integrated paper-based microfluidic system has been developed for influenza A detection with smartphone at point-of-care (POC) settings. The low-cost paper-based microfluidic chip consists of a reagent storage and reaction modules. The storage module, which consists of a couple of reagent chambers with dispensation channels, is responsible for reagent storage and release. The reaction module consists of an absorbent pad and a nitrocellulose (NC) membrane which is functionalized with specific monoclonal antibodies on a test and control spots for immunoassay detection. Microfluidic Dot-ELISA is performed when the dispensed reagent flows through the NC membrane at a controllable speed and reaches the absorbent pad because of the gravity and capillary force without active pumping. A smartphone is used to capture image from the NC membrane with its own camera and process the image with an intelligent algorithm of custom application software which is developed with Java. With a smartphone, the detection result can be displayed and transmitted to other medical agencies if necessary. Experimental results show that, compared with the traditional methods, more convenient and efficient influenza A detection can be achieved with the developed paper-based POC microfluidic chip with the assistance of smartphone.     

Microfluidic systems for extracting nucleic acids for DNA and RNA analysis

This paper is to review the differences in the developments of microfluidic chips for extracting genomic deoxyribonucleic acid (DNA) and viral ribonucleic acid (RNA) from blood by the Biosensor Focus Interest Group (BFIG) in Singapore. DNA was extracted in a multi-step process by isolating and lysing white blood cells (WBC), typically ∼10 μm in diameter. Viral RNA was extracted directly from the submicron viruses in the blood. In terms of basic microfluidic components required, both DNA and RNA extractions used similar mixers for mixing reagents, filters for capturing or separating the blood cells, and a binder for capturing and purifying the DNA/RNA molecules. The designs of the filters were adapted to either capture WBC for DNA isolation or capture all virus particles for RNA isolation. The designs of these two kinds of filters had to be different. Besides the differences in the sizes of WBC and viruses, the concentration of the virus particles is usually much lower than WBC. Thus, a much higher volume of blood for filtering would be required for extracting viral RNA, especially for the intention to detect the viruses at early onset of infection. With proper modifications of the protocols, it has been demonstrated that both genomics DNA and viral RNA could be extracted successfully in these microfluidic chips. The quality of the extracted samples was verified by polymerase chain reaction (PCR) and gel-electrophoresis after the extractions.     

An integrated microfluidic system for live bacteria detection from human joint fluid samples by using ethidium monoazide and loop-mediated isothermal amplification

Periprosthetic joint infection (PJI) is one of the severe complications of prosthetic joint replacement. Delayed PJI diagnosis may anchor bacteria in periprosthetic tissues, and removal of the prosthesis might be inevitable. The diagnosis of PJI depends on the identification of microorganisms by standard microbiological cultures or more advanced molecular diagnostic methods for detection of bacterial genes. However, these methods are relatively time-consuming, labor-intensive and not human error-free. Moreover, it is challenging to distinguish live from dead bacteria by using DNA-based molecular diagnostics since bacterial DNA will be remained in the tissue even after the death of the bacteria. In this work, an integrated microfluidic system has been developed to perform the entire molecular diagnostic process for the PJI diagnosis in a single chip. We combined the loop-mediated isothermal amplification (LAMP) with ethidium monoazide (EMA) in an integrated microfluidic system to identify live bacteria with reasonable sensitivity and high specificity. All the diagnostic processes including bacteria isolation, cell lysis, DNA amplification and optical detection can be automatically performed on the integrated microfluidic system by using a compact custom-made control system. The integrated system can accommodate four primers complementary to six regions of the target genes and improve the detection limit by using LAMP. The limit of detection in this multiple EMA-LAMP assay could be as low as 5 fg/reaction (~1 CFU/reaction) when choosing an optimized primer set as we demonstrated in mecA gene detection. Thus, the developed system for PJI diagnosis has great potential to become a point-of-care device.     

An integrated microfluidic system for fast, automatic detection of C-reactive protein

C-reactive protein (CRP) is a well-known inflammation marker in human beings. This study reports a new microfluidic system for fast, automatic detection of CRP. It contains pneumatic micropumps, a vortex-type micromixer, a pneumatic micro-injector and several microvalves to automatically perform the entire protocol for CRP detection. This includes sample/reagent transportation, incubation between the target CRP and a CRP-specific aptamer, washing processes, and the chemiluminescence development process. In addition, the chemiluminescence signal is measured by using a custom-made optical system which consists of a photomultiplier tube, a portable air compressor and eight electronic magnet valves to quantify the concentration of CRP. When compared to previous works, not only can this new microfluidic system automatically perform the entire process via a new integrated micro-injector and new micropumps, but a new CRP-specific DNA aptamer with a higher affinity and specificity is also used for CRP measurement. Experimental data show that the developed system can automatically complete the entire protocol within 30 min with a detection limit of 0.0125 mg/L, which is superior to previous published results. Moreover, this study also measures CRP concentration from clinical samples to verify the performance of the developed microfluidic system. The results indicate that the measured CRP concentrations from human serums are consistent with those using a benchtop system. The developed system can also detect CRP concentrations from human whole blood without any external sample pretreatment process. This microfluidic system may be promising for point-of-care applications for CRP detection in the future.     

Utilization of nanoparticles in microfluidic systems for optical detection

An increasing interest has been shown in microfluidic systems due to their properties including low consumption of reagents, short analysis time and easy integration. However, despite of these advantages over conventional methods, some limitations in sensitivity and selectivity still exist in microfluidic systems. Recently advancements in nanotechnology offer some new approaches for the detection of target analytes with high sensitivity and selectivity. As a result, it is an appropriate method to enhance the detection sensitivity through a combination between microfluidic system and nanotechnology. Optical detection is a dominant technique in microfluidics because of its noninvasive nature and easy coupling. Numerous studies that integrate optical microfluidic system with nanotechnology have been reported in recent years. Therefore, optical microfluidic systems in combination with nanomaterials (NMs) are reviewed in our work. We illustrate the functions of different NMs in optical microfluidic systems and the efforts of different researchers to improve the performance of devices. After the introduction of different nanoparticle-based optical detection methods, challenges and future directions in the development of nanoparticle-based optical detection schemes in microfluidics have also been discussed.     

Integrated microfluidic preconcentrator and immunobiosensor

We present a microfluidic biosensor that integrates membrane-based preconcentration with fluorescence detection. The concentration membrane was fabricated in polyacrylamide by an in situ photopolymerization technique at the junction of glass microchannels. Liposomes entrapping sulforhodamine B dye molecules were used for signal amplification. The biotin–streptavidin binding system was a model system for evaluating device performance. Biotinylated liposomes were preconcentrated at the membrane by applying an electric field across the membrane. The electric field causes the liposomes to migrate toward the membrane where they are concentrated by a sieving effect. Two orders of magnitude concentration was achieved after applying the electric field for only 2 min. The concentrated bolus was then eluted toward the detection unit, where the biotinylated liposomes were captured by immobilized streptavidin. The integrated system with the preconcentration module shows a 14-fold improvement in signal as opposed to a system that does not include preconcentration.     

An automated microfluidic colourimetric sensor applied in situ to determine nitrite concentration

A stand-alone sensor system with integrated sub-systems is demonstrated. The system is portable and capable of in situ reagent-based nutrient analysis. The system is based on a low cost optical detection method, together with an automated microfluidic delivery system that is able to detect nitrite with a limit of detection (LOD) of 15 nM. The sensor was operated in situ at Southampton Dockhead for 57 h (December 2010) and 375 measurements were taken.     

采用DVD光学头的微流细胞计的细胞分析

一、引言流动血细胞计是当细胞通过盛满液流的设备时,单个生物细胞的物理或者生化特征被检测出来的一个系统。这些系统往往基于光学或者阻抗的测量方法,其中光学流动细胞计是最重要的商业化产品。物理参数如细胞密度或者单个细胞尺寸可以通过基于直流/低频交流阻抗测试法或者高频信号的库尔特计数器(Coulter Counter)获取。使用阻抗频谱参数仪,膜电容和细胞浆电导率等可以在细胞流经时获取。     

An integrated microfluidic system for counting of CD4+/CD8+ T lymphocytes

This study reports on an integrated microfluidic system capable of counting CD4⁺/CD8⁺ T lymphocytes from a whole blood sample, which may be further applied for the rapid screening of the human immunodeficiency virus (HIV) infection. This system is composed of a sample incubation module for fluorescence-labeling of the target cells and a micro-fabricated flow cytometry module for cell counting. First, a pneumatically driven, vortex-type micro-mixer has been adopted for the fluorescence-labeling of CD4⁺/CD8⁺ T lymphocytes from whole blood. After the labeling process, different laser-excited fluorescent signals are detected and are used for counting of CD4⁺/CD8⁺ T lymphocytes as they pass through the detection region of the microflow cytometer. A concentration of 963 cells/μl is counted for cultured CD4⁺ T lymphocytes with a reference concentration of 1000 cells/μl. The ratio of CD4⁺/CD8⁺ T lymphocytes is then calculated. Experimental results show that the results from the microsystem are in agreement with the ones from large-scale flow cytometers. In addition, the entire diagnostic procedure, including the sample incubation and the cell counting, can be automatically performed within 35 min. Therefore, this may become a powerful tool for further biomedical applications, especially for fast screening of HIV infection.     

Non-destructive on-chip imaging flow cell-sorting system for on-chip cellomics

We have developed a non-destructive imaging flow cell-sorting system using an ultra-high-speed camera (shutter speed of 1/10,000 s) with a real-time image analysis unit and a poly(methyl methacrylate) (PMMA)-based disposable microfluidic chip for single-cell-based on-chip cellomics. It has a 3-D micropipetting device that supports fully automated sorting and collection of samples. The entire fluidic system is implemented in a disposable plastic chip, enabling biological samples to be lined up in a laminar flow using hydrodynamic focusing. Its optical system enables direct observation-based cell identification using specific image indexes and phase-contrast/fluorescence microscopy, real-time image processing. It has a non-destructive, wider dynamic range, sorting procedure using mild electrostatic force in a laminar flow; agarose gel electrodes are used to prevent electrode loss and electrolysis bubble formation. The microreservoir used for recultivating collected target cells is contamination-free. An integrated ultra-high-speed droplet polymerase chain reaction measurement module is used for DNA/mRNA analysis of the collected target cells. This system was used to separate cardiomyocyte cells from a mixture of various cells. All the operations were automated using the 3-D micropipetting device. The results demonstrate that this imaging flow cell-sorting system is practically applicable for biological research and clinical diagnosis.     

Modeling and optimization of a multi-enzyme electrokinetically driven multiplexed microchip for simultaneous detection of sugars

A model-based methodology was developed to optimize microfluidic chips for the simultaneous enzymatic quantification of sucrose, d-glucose and d-fructose in a single microfluidic channel with an integrated optical detection system. The assays were based on measuring the change in concentration of the reaction product NADH, which is stoichiometrically related to the concentration of those components via cascade of specific enzymatic reactions. A reduced order mathematical model that combines species transport, enzyme reaction, and electrokinetic bulk flow was developed to describe the operation of the microfluidic device. Using this model, the device was optimized to minimize sensor response time and maximize signal output by manipulating the process conditions such as sample and reagent volume and flow rate. According to this simulation study, all sugars were quantified within 2.5 min in the optimized microchip. A parallel implementation of the assays can further improve the throughput. In addition, the amount of consumed reagents was drastically reduced compared to microplate format assays. The methodology is generic and can easily be adapted to other enzymatic microfluidic chips.     

The determination of phosphorus in a microfluidic manifold demonstrating long-term reagent lifetime and chemical stability utilising a colorimetric method

The chemical stability of the vanadomolybdophosphoric acid method for phosphate determination in a microfluidic manifold is described. The reagent lifetime has been shown to extend to more than 1 year. A stopped flow regime has been implemented, which enabled a very simple microfluidic manifold design to be employed, and has the added advantages of low reagent consumption coupled with less waste generation and access to the complete reaction profile in the optical cuvette on the microfluidic chip. Optical detection was achieved with an UV-LED, integrated into the microfluidic chip holder, coupled to a portable spectrometer via an optical fibre. The manifold includes integration of reagent and sample introduction inlets, a mixing channel and an optical cuvette of 400 μm path length. Two reagent batches were prepared (December 1999 and April 2001) and were shown to still be highly comparable after 1 year in storage. Multiple calibrations have been performed on the microfluidic system over a 12-month period showing only minimal loss in performance and a standard orthophosphate-containing sample was analysed in the microfluidic manifold on a weekly basis with a relative standard deviation of <2.3%.     

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.