Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Microflow Cytometer <s> Abstract This paper presents a novel microfabrication technology of three-dimensional (3D) microstructures with inclined/rotated UV lithography using negative thick photoresist, SU-8. In exposure process, a photomask and a SU-8 coated substrate are fixed together, and tilted or tilted and rotated to a UV source. The reflected UV at the interface between the resist and the substrate is also exploited as well as the incident UV. With the 3D microfabrication technology, various 3D microstructures are easily fabricated such as oblique cylinders, embedded channels, bridges, V-grooves, truncated cones, and so on. The angles between fabricated structures and normal lines are 19.5°, when the incident angle of UV in air is 32°. Thus, the refractive index of SU-8 and the maximum refractive angle at the interface between SU-8 and air are expected about 1.6 and 39°, respectively. <s> BIB001 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Microflow Cytometer <s> The manipulation of fluids in channels with dimensions of tens of micrometres--microfluidics--has emerged as a distinct new field. Microfluidics has the potential to influence subject areas from chemical synthesis and biological analysis to optics and information technology. But the field is still at an early stage of development. Even as the basic science and technological demonstrations develop, other problems must be addressed: choosing and focusing on initial applications, and developing strategies to complete the cycle of development, including commercialization. The solutions to these problems will require imagination and ingenuity. <s> BIB002 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Microflow Cytometer <s> Microfluidics is an emerging field that has given rise to a large number of scientific and technological developments over the last few years. This review reports on the use of various materials, such as silicon, glass and polymers, and their related technologies for the manufacturing of simple microchannels and complex systems. It also presents the main application fields concerned with the different technologies and the most significant results reported by academic and industrial teams. Finally, it demonstrates the advantage of developing approaches for associating polymer technologies for manufacturing of fluidic elements with integration of active or sensitive elements, particularly silicon devices. <s> BIB003 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Microflow Cytometer <s> This review describes recently emerging optical and microfluidic technologies suitable for point-of-care genetic analysis systems. Such systems must rapidly detect hundreds of mutations from biological samples with low DNA concentration. We review optical technologies delivering multiplex sensitivity and compatible with lab-on-chip integration for both tagged and non-tagged optical detection, identifying significant source and detector technology emerging from telecommunications technology. We highlight the potential for improved hybridization efficiency through careful microfluidic design and outline some novel enhancement approaches using target molecule confinement. Optimization of fluidic parameters such as flow rate, channel height and time facilitates enhanced hybridization efficiency and consequently detection performance as compared with conventional assay formats (e.g. microwell plates). We highlight lab-on-chip implementations with integrated microfluidic control for "sample-to-answer" systems where molecular biology protocols to realize detection of target DNA sequences from whole blood are required. We also review relevant technology approaches to optofluidic integration, and highlight the issue of biomolecule compatibility. Key areas in the development of an integrated optofluidic system for DNA hybridization are optical/fluidic integration and the impact on biomolecules immobilized within the system. A wide range of technology platforms have been advanced for detection, quantification and other forms of characterization of a range of biomolecules (e.g. RNA, DNA, protein and whole cell). Owing to the very different requirements for sample preparation, manipulation and detection of the different types of biomolecules, this review is focused primarily on DNA-DNA interactions in the context of point-of-care analysis systems. <s> BIB004 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Microflow Cytometer <s> The aim of microfluidic mixing is to achieve a thorough and rapid mixing of multiple samples in microscale devices. In such devices, sample mixing is essentially achieved by enhancing the diffusion effect between the different species flows. Broadly speaking, microfluidic mixing schemes can be categorized as either "active", where an external energy force is applied to perturb the sample species, or "passive", where the contact area and contact time of the species samples are increased through specially-designed microchannel configurations. Many mixers have been proposed to facilitate this task over the past 10 years. Accordingly, this paper commences by providing a high level overview of the field of microfluidic mixing devices before describing some of the more significant proposals for active and passive mixers. <s> BIB005 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Microflow Cytometer <s> Analysis of the intrinsic fluorescence profiles of individual marine algae can be used in general classification of organisms based on cell size and fluorescence properties. We describe the design and fabrication of a Microflow Cytometer on a chip for characterization of phytoplankton. The Microflow Cytometer measured distinct side scatter and fluorescence properties of Synechococcus sp., Nitzschia d., and Thalassiosira p.; measurements were confirmed using the benchtop Accuri C6 flow cytometer. The Microflow Cytometer proved sensitive enough to detect and characterize picoplankton with diameter approximately 1 μm and larger phytoplankton of up to 80 μm in length. The wide range in size discrimination coupled with detection of intrinsic fluorescent pigments suggests that this Microflow Cytometer will be able to distinguish different populations of phytoplankton on unmanned underwater vehicles. <s> BIB006 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Microflow Cytometer <s> A modern flow cytometer can analyze and sort particles on a one by one basis at rates of 50,000 particles per second. Flow cytometers can also measure as many as 17 channels of fluorescence, several angles of scattered light, and other non-optical parameters such as particle impedance. More specialized flow cytometers can provide even greater analysis power, such as single molecule detection, imaging, and full spectral collection, at reduced rates. These capabilities have made flow cytometers an invaluable tool for numerous applications including cellular immunophenotyping, CD4+ T-cell counting, multiplex microsphere analysis, high-throughput screening, and rare cell analysis and sorting. Many bio-analytical techniques have been influenced by the advent of microfluidics as a component in analytical tools and flow cytometry is no exception. Here we detail the functions and uses of a modern flow cytometer, review the recent and historical contributions of microfluidics and microfabricated devices to field of flow cytometry, examine current application areas, and suggest opportunities for the synergistic application of microfabrication approaches to modern flow cytometry. <s> BIB007 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Microflow Cytometer <s> Stem cells are a powerful resource for producing a variety of cell types with utility in clinically associated applications, including preclinical drug screening and development, disease and developmental modeling, and regenerative medicine. Regardless of the type of stem cell, substantial barriers to clinical translation still exist and must be overcome to realize full clinical potential. These barriers span processes including cell isolation, expansion, and differentiation; purification, quality control, and therapeutic efficacy and safety; and the economic viability of bioprocesses for production of functional cell products. Microfluidic systems have been developed for a myriad of biological applications and have the intrinsic capability of controlling and interrogating the cellular microenvironment with unrivalled precision; therefore, they have particular relevance to overcoming such barriers to translation. Development of microfluidic technologies increasingly utilizes stem cells, addresses stem cell-relevant biological phenomena, and aligns capabilities with translational challenges and goals. In this concise review, we describe how microfluidic technologies can contribute to the translation of stem cell research outcomes, and we provide an update on innovative research efforts in this area. This timely convergence of stem cell translational challenges and microfluidic capabilities means that there is now an opportunity for both disciplines to benefit from increased interaction. <s> BIB008 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Microflow Cytometer <s> Conventional flow cytometry (FC) suffers from the diffraction limit for the detection of nanoparticles smaller than 100 nm, whereas traditional total internal reflection (TIR) microscopy can only detect few samples near the solid-liquid interface mostly in static states. Here we demonstrate a novel on-chip optofluidic technique using evanescent wave sensing for single nanoparticle real time detection by combining hydrodynamic focusing and TIR using immiscible flows. The immiscibility of the high-index sheath flow and the low-index core flow naturally generate a smooth, flat and step-index interface that is ideal for the TIR effect, whose evanescent field can penetrate the full width of the core flow. Hydrodynamic focusing can focus on all the nanoparticles in the extreme centre of the core flow with a width smaller than 1 μm. This technique enables us to illuminate every single sample in the running core flow by the evanescent field, leaving none unaffected. Moreover, it works well for samples much smaller than the diffraction limit. We have successfully demonstrated the scattering imaging and counting of 50 nm and 100 nm Au nanoparticles and also the fluorescence imaging and counting of 200 nm beads. The effective counting speeds are estimated as 1500, 2300 and 2000 particles per second for the three types of nanoparticles, respectively. The optical scattering spectra were also measured to determine the size of individual Au nanoparticles. This provides a new technique to detect nanoparticles and we foresee its application in the detection of molecules for biomedical analyses. <s> BIB009
Although conventional flow cytometers have gained profound success in cell sorting and analysis, they are bulky, demand large amounts of expensive reagents with complicated processing steps, are complicated in manipulation and require high maintenance costs. Typically, a flow cytometer will be located in a single facility where many hundreds of users will need access to it. These limitations restrict their uses in POC diagnostics, in situ pathogen monitoring and other application where portability, handling small volume samples, low operation costs and ease of operation are essential. Owing to the recent development of lab-on-chip (LOC) technology, microfabrication and micromachining techniques, the miniaturization of a flow cytometer can be achieved. The microfabrication of a flow cytometer with 3D microstructures can be accomplished by 3D microfabrication techniques utilizing UV lithography BIB001 . Microfluidic mixing BIB005 , microfluidic cell sorting and the miniaturization of pumps and valves BIB003 provide a basic foundation for miniaturizing the fluidic handling to develop a microchip-based flow cytometer. Researchers have been able to take advantage of these advanced microfabrication technologies to miniaturize a flow cytometer to a microscale or even a nanoscale platform. Controlling fluids in a microchannel allows microchip-based flow cytometer to be applied in POC diagnostics and lab-on-a-chip devices offers unique advantages BIB002 , such as reducing the volumes of reagents, shortening the turnaround time between inspection and results and lowering the associated costs BIB004 . To date, the throughput of microflow cytometers can reach up to 50,000 cells/s BIB007 , allowing microflow cytometers to have many applications: Titmarsh et al. BIB008 discussed how microfluidic technology spurred on the development of stem cell-derived therapies. Hashemi et al. BIB006 successfully distinguished different populations of phytoplankton with high sensitivity by measuring light scatter and fluorescence properties by a microflow cytometer. More demonstrations on diagnostic and point-of-care applications have been addressed in recent review papers BIB004  . A novel optofluidic device-based microflow cytometer emerged recently. Optical components and novel liquid lenses are used in microfluidic devices. Liquid-core/liquid-cladding waveguides and liquid core/air-cladding lens systems with larger refractive index contrast lead to less propagation losses and resulted in better optical confinement  . Additionally, an integrated on-chip lens system or grooved on-chip fibers further reduce the size of the microflow cytometer. Built-in waveguides also are free of optical alignment, making operation much easier. Recently, Liang et al. took advantage of evanescent waves present at the liquid-liquid interface of immiscible flows to count the nanoparticles on an optofluidic microchip BIB009 .