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(a) Continuous flow rates at 1, 4, 7 and 10 L/min at room temperature while keeping time constant at 2 min period. The concentration of target DNA is 50 pM and the height of microfluidic flow channel is 50 m. Fluorescence signals in the first row correspond to 10 L/min, 7 L/min in the second row, 4 L/min in the third row and 1 L/min in last row. Note that there is no indication of binding at the Corona Virus DNA sites, the control or inside the ellipses. (b) Fluorescence intensity vs. continuous flow at different heights for 2 min. Flow rate of continuous flow were 1, 4, 7 and 10 L/min. The concentration of target DNA was 50 pM. The number of experiments done (n) is equal to 6.
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In this investigation we report on the influence of volumetric flow rate, flow velocity, complementary DNA concentration, height of a microfluidic flow channel and time on DNA hybridization kinetics. A syringe pump was used to drive Cy3-labeled target DNA through a polydimethylsiloxane (PDMS) microfluidic flow channel to hybridize with immobilized...
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... a continuous flow regime to optimize the DNA hybridization speed and lower detection limit. We also investigated the hybridization speed for a fixed amount of time (hopefully as short as possible) at various flow rates. To study the influence of various flow rates at a fixed time of 2 min, hybridization of 10, 7, 4 and 1 L/min were compared (see Fig. 9a). The experiments were carried out at a fixed concentration of 50 pM of Cy3-labeled target DNA. The results show that the West Nile Virus DNA capture probes have a greater chance to hybridize with Cy3-labeled target DNA at a higher flow rate (10 L/min versus 1 L/min), and at a lower height (18 m versus 50 m) at a duration of 2 min (see ...
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... Fig. 9a). The experiments were carried out at a fixed concentration of 50 pM of Cy3-labeled target DNA. The results show that the West Nile Virus DNA capture probes have a greater chance to hybridize with Cy3-labeled target DNA at a higher flow rate (10 L/min versus 1 L/min), and at a lower height (18 m versus 50 m) at a duration of 2 min (see Fig. 9b). The reason can be attributed to a larger number of target DNA molecules being delivered into the microfluidic flow channel at larger flow rates when time is held constant. Secondly, the total diffusion distance of target DNA molecules to capture probes is decreased when the channel height is reduced. We summarize all the results ...
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... a continuous flow regime to optimize the DNA hybridization speed and lower detection limit. We also investigated the hybridization speed for a fixed amount of time (hopefully as short as possible) at various flow rates. To study the influence of various flow rates at a fixed time of 2 min, hybridization of 10, 7, 4 and 1 L/min were compared (see Fig. 9a). The experiments were carried out at a fixed concentration of 50 pM of Cy3-labeled target DNA. The results show that the West Nile Virus DNA capture probes have a greater chance to hybridize with Cy3-labeled target DNA at a higher flow rate (10 L/min versus 1 L/min), and at a lower height (18 m versus 50 m) at a duration of 2 min (see ...
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... Fig. 9a). The experiments were carried out at a fixed concentration of 50 pM of Cy3-labeled target DNA. The results show that the West Nile Virus DNA capture probes have a greater chance to hybridize with Cy3-labeled target DNA at a higher flow rate (10 L/min versus 1 L/min), and at a lower height (18 m versus 50 m) at a duration of 2 min (see Fig. 9b). The reason can be attributed to a larger number of target DNA molecules being delivered into the microfluidic flow channel at larger flow rates when time is held constant. Secondly, the total diffusion distance of target DNA molecules to capture probes is decreased when the channel height is reduced. We summarize all the results ...
Citations
... Since no extra instruments and treatment are required for bonding and debonding, this method has been frequently applied to reversibly bond PDMS substrates to glass plates. [42][43][44][45] The bonded layers with the self-adhesive property of PDMS material can be easily detached by hands; 46 however, the bonding strength of this method is weak, and the maximum reported bonding strength with this method is around 22 kPa. 47 In addition, TPE (Thermoplastic Elastomers) materials have also been applied to reversible bonding with self-adhesive properties, 48,49 and the reported maximum working pressure is 3.4 times higher than that of PDMS cases. ...
With the development of microfluidic technology, new materials and fabrication methods have been constantly invented in the field of microfluidics. Bonding is one of the key steps for the fabrication of enclosed-channel microfluidic chips, which have been extensively explored by researchers globally. The main purpose of bonding is to seal/enclose fabricated microchannels for subsequent fluid manipulations. Conventional bonding methods are usually irreversible, and the forced detachment of the substrate and cover plate may lead to structural damage to the chip. Some of the current microfluidic applications require reversible bonding to reuse the chip or retrieve the contents inside the chip. Therefore, it is essential to develop reversible bonding methods to meet the requirements of various applications. This review introduces the most recent developments in reversible bonding methods in microfluidics and their corresponding applications. Finally, the perspective and outlook of reversible bonding technology were discussed in this review.
... For example, Jacoc Glanville, Ph.D. works with the SARS-CoV sequences using DNA shuffling, and he is looking for a variant that achieves blocking effects on 2019-nCoV. There is also the design of synthetic peptides or aptamers (Haußner, Lach & Eichler, 2017) that can induce an immune response mediated by CD4+ and CD8+ cells through an epitope-parotope interaction (Bojin, Gavriliuc, Margineanu & Paunescu, 2020); or nano-vaccines based on nanoparticles such as VLPs (virus-like particles) acting as delivery vehicles of the antigens that trigger protective immunity by inducing the generation of antibodies to fight the virus (Palestino, García-Silva, González-Ortega & Rosales-Mendoza, 2020); or a plasmid with synthetic DNA of the coronavirus S protein, as was done with MERS-CoV (Muthumani et al., 2015); or the application of new diagnostic techniques such as microfluidics, which were used for the SARS-CoV virus (Kim, Marafie, Jia, Zoval & Madou, 2006;Zhou et al., 2004). All these treatments promise to be more effective than the use of angiotensin 1 receptor blockers (AT1R) and ACE. ...
Synthetic biology aims to develop cells with entirely new functions not found in nature. These functions are manifested through pathways created of genes from other microorganisms linked by molecular techniques such as Bio-Brick Assembly. Some of these linkages can adopt a Boolean behavior and generate what is known as a genetic circuit that is mainly composed of the functional parts of a gene (Promoter, RBS, ORF, Terminator). These interchangeable parts form what is called a Bio-Brick, which can act as a logical gateway by showing an excellent stability and the activation of genetic memory that can lasts after several generations. As a result of the different types of behavior that Bio-Bricks present, they are highly attractive for the industry because it would be enough to choose the best type of circuit for multiple applications both in the biomedical industry (cancer drugs, malaria, specific antibodies, microbiome engineering) or the energy industry in order to produce second-generation biofuels that can compete effectively with fossil fuels; it has also been discovered that due to its usefulness in different fields, it represents a solution to problems such as high greenhouse gas emissions or the current pandemic caused by the appearance of the SARS-CoV-2 virus. Biología sintética: Sobre el desarrollo de circuitos genéticos y su aplicación en biociencias y en biocombustibles resumen La biología sintética tiene como objetivo desarrollar células con funciones totalmente nuevas y que no se encuentran en su naturaleza. Estas funciones se manifiestan a través de rutas creadas a partir de genes provenientes de otros microorganismos vinculados por técnicas moleculares como el ensamblaje de Bio-Brick. Algunos de estos enlaces pueden adoptar un comportamiento booleano y generar lo que se conoce como un circuito genético, compuesto principalmente por las partes funcionales de un gen (Promotor, RBS, ORF, Terminador). Estas piezas intercambiables forman lo que se conoce como Bio-Brick, que actúa como una puerta lógica al mostrar una estabilidad excelente y una memoria genética que perdura después de varias generaciones. Por los distintos tipos de comportamiento que presentan los Bio-Bricks, son muy atractivos para la industria, ya que con solo elegir un tipo de circuito sus aplicaciones son diversas, por ejemplo en la industria biomédica para (medicamentos contra el cáncer, la malaria, anticuerpos específicos e ingeniería del microbioma), o en la industria energética para (generar biocombustibles de segunda generación que compitan eficazmente con los combustibles fósiles; también se ha descubierto que por su utilidad en diferentes ámbitos, representa una solución a problemas como las altas emisiones de gases de efecto invernadero o la actual pandemia provocada por la aparición del SARS-CoV-2. Palabras clave: biología sintética, circuitos genéticos, Bio-Bricks, ingeniería del microbioma, rutas sintéticas, producción de biocombustibles. Artículo recibido el 15 de septiembre del 2021. Artículo aceptado el 10 de marzo del 2023. ARTÍCULO DE REVISIÓN
... For example, Jacoc Glanville, Ph.D. works with the SARS-CoV sequences using DNA shuffling, and he is looking for a variant that achieves blocking effects on 2019-nCoV. There is also the design of synthetic peptides or aptamers (Haußner, Lach & Eichler, 2017) that can induce an immune response mediated by CD4+ and CD8+ cells through an epitope-parotope interaction (Bojin, Gavriliuc, Margineanu & Paunescu, 2020); or nano-vaccines based on nanoparticles such as VLPs (virus-like particles) acting as delivery vehicles of the antigens that trigger protective immunity by inducing the generation of antibodies to fight the virus (Palestino, García-Silva, González-Ortega & Rosales-Mendoza, 2020); or a plasmid with synthetic DNA of the coronavirus S protein, as was done with MERS-CoV (Muthumani et al., 2015); or the application of new diagnostic techniques such as microfluidics, which were used for the SARS-CoV virus (Kim, Marafie, Jia, Zoval & Madou, 2006;Zhou et al., 2004). All these treatments promise to be more effective than the use of angiotensin 1 receptor blockers (AT1R) and ACE. ...
Synthetic biology aims to develop cells with completely new functions that are not found in their nature. These functions can be achieved through pathways created from genes from other microorganisms linked by molecular techniques such as Bio-Brick Assembly. Some of these links can adopt a Boolean behavior and generate what is known as a genetic circuit, which is composed mainly of the functional parts of a gene (Promoter, RBS, ORF, Terminator), and together they form something called Bio-Brick that can act as a logical gateway and show characteristics such as greater stability and activation memory, even after several generations. In this way they can have highly attractive behaviors for the industry, because it would be enough to choose the best type of circuit for multiple applications, both in the biomedical industry (cancer drugs, malaria, specific antibodies, microbiome engineering), and the energy industry, as second generation biofuels that can compete effectively with fossil fuels. Among many other applications that are expected to address current problems that threaten our lifestyle globally such as high greenhouse gas emissions or the current 2019-nCoV pandemic by the SARS-CoV-2 virus.
... To overcome the requirement of large amount of costly DNA samples, the electro-kinetic micro/ nanofluidic technologies [8][9][10] offer many advantages including fast mixing/reaction rate, improved sensitivity, easy automation and real time monitoring. Even though the existing channel-microfluidic systems [11][12][13][14] are widely employed for DNA hybridization investigations, the redundant supporting equipment (valves, pumps or tubes) and risk of valve clogging still confine the system flexibilities for different diagnostic applications. ...
DNA hybridization kinetics has been playing a critical role in molecular diagnostics for binding discrimination, but its study on digital microfluidic (DMF) systems is ultimately restrained by the laminar flow condition. The kinetic mixing technique is widely employed to ensure a fast reaction rate, but poses intrinsic risk in cross contamination and exhibits instable fluorescence intensity during the droplet transportation. While the electrothermal technique can provide stationary droplet mixing through the established thermal gradient within the hybridization solution, the significant increase in the droplet temperature will inevitably undermine the hybridization equilibrium and jeopardize the binding discrimination. To enhance the hybridization efficiency while ensuring a stable droplet temperature (within ±0.1℃), this paper presents a DMF platform that can perform isothermal hydrodynamic-flow-enhanced droplet mixing. Specifically, with a single electrode, droplet-boundary oscillation under a slow AC actuation is studied for improving the reaction rate. The dependencies between the mixing efficiency and the actuation voltage, actuation frequency and the spacer thickness are also systematically studied. Reliable mixing efficiency improvement is further validated over a wide range of solute concentrations. The results from real-time on-chip DNA hybridization kinetics with stationary droplets using the complete sandwiched DMF system shows that the proposed rapid mixer can achieve the same hybridization equilibrium with >13 times faster reaction rate when compared to the reference one through pure diffusion, while preventing biased hybridization kinetics as demonstrated in the electrothermal technique.
... Reduction of the channel height combined with higher flow rate enhances the mass transport of the target DNA to the surface immobilized probes and increases the hybridization signal. Compared to passive hybridization, the hybridization of DNA in a microfluidic channel generates higher signal intensities at lower concentration of the target DNA [5]. ...
... Future improvements to the experimental parameters of the nucleic acid hybridization system will include modification of the channel height and buffer flow rates. Higher flow rate and lower channel height are reported to increase the hybridization efficiency and reduce the detection time because diffusive transport plays a more significant role in the hybridization process in a microfluidic device [5]. ...
... 7 However, the simplest and most efficient way to decrease the reaction time is to further shrink the channel dimensions in order to bring target molecules closer to the surface immobilized probes, providing that molecular transport through the channel is assisted by convection. 8 Lately, we have witnessed the rapid development of nanofluidics, allowing biomolecular manipulation and observation down to the single molecule level. [9][10][11][12][13][14][15][16][17][18][19] While most applications of nanochannels have taken advantage of either the confined fluidic environment for biophysics studies and single molecule separation/analysis or the influence of the electrical double layer to create ion depletion zones for molecular sieving or concentration, there have been surprisingly few studies a) Authors to whom correspondence should be addressed. ...
We propose biofunctionalized nanofluidic slits (nanoslits) as an effective platform for real-time fluorescence-based biosensing in a reaction-limited regime with optimized target capture efficiency. This is achieved by the drastic reduction of the diffusion length, thereby a boosted collision frequency between the target analytes and the sensor, and the size reduction of the sensing element down to the channel height comparable to the depletion layer caused by the reaction. Hybridization experiments conducted in DNA-functionalized nanoslits demonstrate the analyte depletion and the wash-free detection ∼10 times faster compared to the best microfluidic sensing platforms. The signal to background fluorescence ratio is drastically increased at lower target concentrations, in favor of low-copy number analyte analysis. Experimental and simulation results further show that biofunctionalized nanoslits provide a simple means to study reaction kinetics at the single-pixel level using conventional fluorescence microscopy with reduced optical depth.
... In addition, electrochemical signals can be directly interfaced with most measurement equipment while other signal modalities may require a transducer to convert the signal (Dukkipati and Pang, 2006; Fang et al., 2009; Pavlovic et al., 2008; Xu et al., 2009). Additional advances have appeared using microfluidics for DNA hybridization (Henry and O′ Sullivan, 2012; Kim et al., 2006; Tai et al., 2013; Xu et al., 2009; Yang et al., 2014) although limitations have likely hampered progress. The most important limitations to overcome include: sample preparation and mixing of fluids (due to the low sample volume and low Reynolds number), physical and chemical effects (including capillary forces, surface roughness, chemical interactions between construction materials and analytes), and low electrochemical signal-to-noise ratio (produced by the reduced surface area and volume) (Beebe et al., 2002; Bhushan, 2010; Ghallab and Badawy, 2010; Mariella, 2008). ...
... Recently, there have been a few advances in incorporating electrochemical sensing in microfluidics for DNA hybridization. Studies have been performed regarding hybridization kinetics 31 and integration of sensors for detection 15 in microfluidic channels. However, there still exists a need for a rapid high throughput microfluidic device that can analyze DNA hybridization events in parallel without complicated sample preparation steps. ...
Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.
... With micromachining techniques, the hybridization microchannels are aligned with rows of probe spots. Both straight [47][48][49][50][51][52][53][54][55] and serpentine microchannels have been designed [23,[56][57][58][59][60]. Wang et al. used a bioarray chip for a mutational analysis (Figure 8.1a and b) [48]. ...
... The reduction of the microchannel height enhanced the mass transport of the target DNA to the capture probes and thus generated higher hybridization signals. Moreover, the DNA molecules were stretched in nanoflows, which offered further advantages for hybridization efficiency [49,50,128]. Thereafter, PCR product samples from the genomic extraction of three fungal pathogens were successfully detected with the CD-like bioarray assembly under optimized conditions [77]. ...
8.1 INTRODUCTION TO NANOFLUIDIC DNA BIOARRAY HYBRIDIZATION Nucleic acid hybridization techniques feature the use of a probe nucleic acid mol-ecule and a target nucleic acid molecule. Here, probe molecules are usually short single-stranded nucleic acids (DNA or RNA) or oligonucleotides with known sequences; whereas target molecules are prepared from polymerase chain reaction (PCR) amplification of genomic extracts. Probe-target hybridization leads to the for-mation of a double-stranded molecule, called a duplex. The method of DNA bioar-ray hybridization evolved from Southern blotting technology based on solid-phase hybridization in the early 1990s [1]. This method relies on the immobilization of the probe molecules onto a solid surface to recognize their complementary DNA target sequences by hybridization. Millions of features have been integrated onto a standard glass or silicon slide by microprinting or in situ synthesis of oligonucle-otides [2,3]. The relative abundance of nucleic acid sequences in the target solu-tion can be measured from chip hybridization results optically, electrochemically, or radiochemically, with proper detection labels [4]. DNA bioarrays have dramatically accelerated many types of investigations including gene expression profiling, com-parative genomic hybridization, protein–DNA interaction studies (chromatin immu-noprecipitation), single-nucleotide polymorphism (SNP) detection, as well as nucleic acid diagnostic applications. The progress of DNA bioarray technology during the last couple of years has been summarized in many books and reviews [4–8]. With the rapid growth of microelectromechanical systems (MEMS), microfluidic/ nanofluidic technology has been developed rapidly with many applications over the CONTENTS
... To provide a perspective to the range of dimensions used in recently published works in CD microfluidic platforms, Table 1 summarizes some of the typical CD microchannel configurations reported along with angular velocities. The width of channels varies from 20 μm (Brenner 2005) to 500 μm Kim et al. 2006) whereas the depth of channels varies from 34 (Madou et al. 2000) to 1,000 μm (Riegger et al. 2006). The variation of lengths is from 8.4 to 21 mm (Ducrée et al. 2005;Brenner 2005). ...
This study investigates the influence of Coriolis force on transport and hybridization of ssDNA molecules in compact disk (CD) microfluidic platform where centrifugal force is used as the driving force. While the effect of Coriolis force on fluid flow in CD microfluidic channels has been studied experimentally and numerically only recently, its influence on ssDNA molecule migration and hybridization has not been investigated so far. This study addresses this phenomenon through numerical simulation and demonstrates that for most practical geometrical configurations and angular velocity ranges reported in the literature, the Coriolis force introduces significant qualitative and quantitative spatial variations in the hybridization of ssDNA molecules, particularly at locations near the periphery. In a particular example investigated here, hybridization was observed to reach steady-state at some locations in about half the time required in the absence of Coriolis force. However, our results further indicate that the time frame for hybridization is so fast (
