A Disposable DNA Analysis Chip with a Powerless Actuator

A non-instrumented, single-use, affordable, and fully- yet safely-disposable DNA analysis system for Point Of Care (POC) diagnostic process has been proposed by integrating (1) a hydration-reactive mixture for a portable heating element as a powerless actuator, (2) commercially available optical adhesive lms as valves, and (3) an exothermic reaction-based recombinant polymerase amplication (RPA) process for non-instrumented DNA amplication. The operational error tolerance of the adhesive valves was evaluated by gas production and long-lasting ability, and the amplication performance of the RPA device was validated by gel electrophoresis. Finally, a DNA analysis device was fabricated and tested based on a hydration reaction with a DNA extraction microuidic channel and an exothermic reaction-based RPA device. In the DNA extraction process, dimethyl adipimidate (DMA) solution was used to eliminate some required injection steps from the extraction process. The integrated system's functionality was successfully demonstrated, and the suggested system could become a foundation for the ultimate total solution for POC DNA analysis.


Introduction
The need for point-of-care (POC) devices has been urged by the emergence of a novel coronavirus, SARS-CoV-2 1 . Micro uidic Lab-on-a-Chip (LOC) technologies have brought revolutionary changes and development in the POC platforms by providing easy-to-use, cost-effective miniaturised systems with faster analysis time [2][3][4] . POC systems have led to effective prevention and early detection of diseases which help deplete mortality drastically [5][6][7] .
Typical POC diagnostic devices, dipstick tests have been widely used for tests of pregnancy, in uenza, cardiac markers and other infectious diseases [7][8][9][10] . This type of POC diagnostic system, however, has limitations to achieve higher accuracy. For enhancing detection accuracy, nucleic acids (DNA and RNA) are widely used as biomarkers to identify speci c infectious diseases [11][12][13] . However, multi-step and complex uid handling is essentially required for the work ow of molecular diagnostics 10 (sample preparation, target ampli cation and signal read-out) 7 .
A micro uidic LOC system is the most suitable platform for POC diagnostic system. Bio-analytical devices integrated with the micro uidic component can be used for multi-step processes in a miniaturised system with fast analysis time, high sensitivity and speci city 14,15 . Especially, micro uidic components based polymeric materials have many advantages, including low-cost fabrication, duplicability and disposability. In a nucleic acids LOC system, the three main components that determine its cost, performance and practicability are the pumps/actuators, valves, and the process of nucleic acid ampli cation.
Micro uidic valves are critical driving components in the LOC system besides pumps and mixers 16,17 .
Suitable micropumping methods for ow control represent a major technical hurdle in developing micro uidic systems for point-of-care testing (POCT). Passive pumping for LOC systems is essential in POC systems 18 to avoid the need for cumbersome external equipment (i.e., syringe pumps and pressure pumps), which are still widely seen in micro uidic devices 19,20 . They control uid ows by opening or closing uidic passageways and are usually actuated by external power resulting in a bulky and complex design and limitation in POC testing applications. Various microvalves have been introduced based on their actuating principles, such as electrochemical, piezoelectric, magnetic, electromagnetic, pneumatic, thermopneumatic, shape memory alloy, surface acoustic wave [21][22][23][24][25][26][27][28] . Also, various kinds of powerless valves have been suggested for micro uidic devices [29][30][31] , together with the development of the powerless pump. Capillary force, gravitational force and nger pressure have been widely used as actuation sources to transport samples in POC diagnostic systems 3,32−34 . Three representative methods have a common problem: they are hard to control the ow rate of reagents and use precise control required reaction steps.
An original polymerase chain reaction (PCR) process was designed to amplify a portion of DNA, an essential step for nucleic acid analysis: temperature control is crucial for the procedure. Due to the commonplace features of the methods, PCR is evolved far beyond simple target DNA or RNA detection.
Fu, Yayun, et al. describes a low-cost and straightforward self-priming compartmentalisation platform for PCR analysis 35 . The critical element of the platform is the degassed PDMS pump aligned with the outlet port of the chip. It creates the negative pressure in the channel, which automatically derives the sample and oil into a microchamber for self-partition. Therefore, the platform doesn't require an external component that needs power for pumping. Salman, Abbas, et al. report a PCR micro uidic device by integrating three systems: micro uidic PCR chip, thermal cycler, and uorescence detector 36 . The thin micro uidic layer of the chip facilitates rapid temperature change, allowing fast cycling times. In addition, the device incorporated with photodetector enables further analysis to monitor the PCR progress. But, both devices are not suitable for the POC diagnostic assays due to time demanding and complex external components that need power, respectively.
In addition, nucleic acid testings such as the reverse transcription-polymerase chain reaction (RT-PCR) have become the most reliable method for COVID-19 detection due to their accuracy and sensitiveness to viral genomes recognised as a gold standard by WHO for virus detection technique. Heating elements, however, is not avoidable due to the thermal cycling process of the PCR. Thus, the method heavily depends on the equipment, well-trained staff, and equipped laboratories 37,38 .
Unlike conventional PCR, isothermal ampli cation methods enable the ampli cation of DNA at one constant temperature. Loop-mediated isothermal ampli cation (LAMP), recombinant polymerase ampli cation (RPA), rolling circle ampli cation (RCA), and helicase dependent ampli cation (HDA) employ the isothermal method. They have been used for DNA-based POC diagnostic applications [39][40][41][42][43][44] . The RPA is one of the popular isothermal methods used in POC testing. Its isothermal ampli cation overcomes shortcomings in temperature control of PCR based tests while providing good sensitivity, lowcost, and fast detection with simple instruments such as paper-based micro uidic devices. It can achieve Page 4/17 RPA method would give more errors and contamination, the reverse-transcription RPA (RT-RPA) was successfully used as an isothermal alternative to RT-PCR for viral disease detection such as Ebola 46 .
Researchers have made enormous efforts to develop non-instrumented nucleic acid ampli cation devices with various isothermal ampli cation methods to realise equipment-free POC diagnostic systems. Liu 47,48 . We believe the non-instrumented RPA device is the most suitable isothermal ampli cation method for POC applications due to the low operating temperature and less time. Therefore, it is worthy of designing a simple powerless controllable pump with a microvalve and non-instrumented RPA device.
This paper presents a non-instrumented DNA analysis system development that enables total analysis of DNA biomarkers from sample to results within 1 hour. This proof-of-concept study employs 1) a selfpowered actuator (or pump) based on a hydration reaction and as a valve; the gas ow is controlled by attaching or detaching a cover lm on the microchannels' holes (inlets and outlets). Then 2) an exothermal RPA that ampli es DNA with self-powered heating. We evaluated the performance of these components individually and demonstrated the integrated non-instrumented DNA analysis system.

Material and Selection of the powder mixture for
Hydration reaction based powerless pump and valve Distilled (DI) water and phosphate-buffered saline (1X PBS) were purchased from Invitrogen (Carlsbad, CA). 3-aminopropyltriethoxysilane (APTES), bovine serum albumin (BSA), dimethyl adipimidate (DMA), sodium bicarbonate, agarose, ethidium bromide (EtBr) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lysis buffer (AL buffer and proteinase K) was purchased from Qiagen (Hilden, Germany). A portable heating element (powder mixture) was purchased from Outing (Seoul, Korea). Rehydration buffer, dried enzyme pellets and magnesium acetate (MgAc) were purchased from TwistDX (Cambridge, UK). Other chemicals were analytical reagent grade and were used as received. All samples and buffers were prepared using DI water and PBS.
A commercially available optical adhesive lm (MicroAmp™, Thermo Fisher Scienti c) was utilised as a valve; manually detaching/attaching the piece on chambers acted as open/close valves to control the ow direction. The idea is creative, affordable, straightforward, yet practical for any general micro uidic system design.
The performance of powerless pumps or actuators is evaluated by how easy the ow rate is, how uniform the ow rate is, and how long the operation lasts. For hydration reaction-based, gas-driven pumps, the composition of the powder mixture, amount of powder and amount of water would directly affect the production rate, duration of gas, operating ow rate, and time of the pumps. In this work, we choose a speci c powder mixture that is used as a portable heating element for ready-to-eat products, and it is composed of aluminium (Al; 47%), calcium hydroxide (CaO; 30%), calcium carbonate (CaCO 3 ; 11%), sodium carbonate (Na 2 CO 3 ; 10%) and sodium hydroxide (NaOH; 2%). The mixture of CaO and Al possibly produces heated gases by reacting with water. CaCO3, Na2CO3 and NaOH play a dominant role during the hydration reaction to increase reaction (operating) time. The same mixture is used for both the hydration reaction and exothermal RPA.

Design and Fabrication of a non-instrumented gasdriven micro uidic system
Metal alloy particles can produce heated gases by reacting with water. The produced gases are potentially used as not only a heat source but also an actuation source. The well-designed composition can make precisely controlled and long-lasting production of gases of metal alloy mixture with other chemical ingredients, and it provides a high-performance powerless micropump. In addition, the gas ow can be controlled by attaching cover lms to or detaching cover lms from holes of a microchannel (inlet or outlet) for the switching of on-off condition and ow direction. concept for a non-instrumented gas-driven micro uidic system. At the beginning of actuator operation, cover lms are detached from the inlet of a microchannel sequentially. Then, water is dropped onto the powder mixture for the hydration reaction using a micro-pipette (i). With the hydration reaction, hydrogen gas is immediately formed. By attaching new cover lm onto the inlet hole, the formed hydrogen gas within the limited space of the microchannel pushes reagents along a microchannel to an outlet (ii).
Based on the principle, we design a powerless gas-driven micro uidic device, as shown in Fig. 1. The powder mixture is put in a deep container located at the top end of the microchannel. Before covering the upper layer of a microchannel, we would place reagents in segmented positions of the microchannel with enough clearances according to the reaction sequence. The chemical treated or receptor immobilised part is attached to the opened region of a reaction zone in the microchannel to perform various bio-analysis processes after covering the upper layer. At the last step for the preparation of the micro uidic device, a hole located in the next above the powder mixture is covered with a cover lm to prevent pre-reaction of powder mixed with water in the air.

Development of Exothermic reaction-based recombinant polymerase ampli cation (RPA) device
The RPA method is one of the isothermal ampli cation methods; especially, it is performed at relatively low temperatures (37 ~ 42°C) during a shorter process time (25 ~ 35 min) without a thermal cycler. In the experiment, the speci c composition of powder mixture in the previous section was used as the solute of exothermic reaction for the heat-up process in the RPA method and water was carried with a Whatman lter paper (Sigma-Aldrich, USA) to the powder container. The RPA device consists of two main parts; the upper reaction chamber and lower container parts, as shown in Fig. 5(a). All layers of the RPA device were fabricated by CO 2 laser cutting/engraving method using PMMA sheets and assembled by attaching double-sided tapes between layers. The container part includes a container layer with two large holes for a powder container (width: 20 mm, length: 20 mm and height: 3 mm) and a water container (10 mm × 20 mm × 3 mm) and a base layer with an engraved groove for a lter paper. In the reaction chamber part, two oated reaction chambers and a wide hole for the water container were deeply engraved and cut through on a reaction chamber layer, respectively. The layer was covered with a thin top layer cut through with four hoes for a gas vent, two reaction chambers and a water container.

Design and Fabrication of a Disposable DNA Analysis Chip with a Powerless Actuator
We designed an integrated system that can perform DNA extraction, ampli cation and read-out without any additional instruments using the powerless hydration reaction-based gas-driven pump and the noninstrumented exothermic reaction-based RPA device. The integrated system, non-instrumented DNA analytical system, consists of four main components: i) reagent reservoir, ii) DNA extraction, iii) DNA ampli cation & detection and iv) container components, as shown in Fig. 2. First, the reagent reservoir component was designed as an elliptical capsule shape of 9 reservoirs (8 reservoirs with 40 µl volume and a reservoir with 30 µl volume) connected in a line for the storage and separation of reagents depending on their purposes. In addition, the air in empty reservoirs acts as spacers between neighbouring reagents to prevent mixing with each other in the sequential injection process. Second, to perform the DNA extraction process (e.g. lysing, washing, elution steps), we designed 4 mixing channels with a spiral shape (1 mm width) and a long binding channel with a meandering shape (1 mm width). In addition, one end outlet of the binding channel was connected with a channel toward a waste container, and the other outlet was connected with a broken channel near reaction chambers. Third, the waste channel and the broken channel were engraved on the top of a reaction chamber layer. As represented in the previous section, three oated reaction chambers with each 60 µl volume for RPA were deeply engraved from the top of the reaction chamber layer. Lastly, a container of powder for an exothermic reaction, a container of water, and a container of powder for hydration reaction with 6 × 6 mm 2 circular areas were designed as the container components.
All of the designed layers with double-sided tapes were cut or engraved by a CO 2 laser machine. From the bottom side, a base layer, a container layer and a reaction chamber layer were fabricated and assembled using 3 mm thickness of PMMA sheets and 0.1 mm thickness of double-sided tapes, respectively. Then, the reaction chamber layer was covered with a double-sided tape and 0.5 mm thickness of a PMMA sheet. For the reagent reservoir component, 1 mm thickness of a PMMA sheet was cut, and it was attached to the cover layer of the reaction chamber with a double-sided tape. To connect the reagent reservoir layer and the DNA extraction layer, an intermediate layer was fabricated using 0.5 mm thickness of a PMMA sheet and attached between the layers with double-sided tapes. On the top of the integrated system, 50 × 24 mm 2 sized glass, functionalised with an amine group, was attached. In addition, four different designs of detachable channels and covers were cut by CO 2 laser cutting/engraving machine using optical adhesive lms (Invitrogen, USA) ( Fig. 2(v)). Finally, the whole system was assembled; double-sided tapes were attached between parts and four detachable channels and covered onto their proper positions.
2.5 Analysis process of DNA from urine sample using the disposable DNA analysis chip with a powerless actuator The DNA analysis process is started from DNA extraction, and it is ended with the detection of ampli ed target DNA through DNA ampli cation. To obtain high-purity DNA, binding DNA extracted from cells using DMA 49 and washing the unbound DNA and other molecules with PBS are necessary for the DNA extraction step. One of the strengths of using DMA as a DNA binding agent is that DMA solution can be mixed with a lysis buffer without inhibition; thus, the solutions can be stored together in a reservoir. As mentioned in the previous section, all reagents used for the DNA analysis are inserted into reservoirs and reaction chambers according to reaction sequences in the fabrication step. Firstly, we placed 11 µl of lysis buffer (10 µl of AL buffer with 1 µl of proteinase K) and 20 µl of 25 mg/ml DMA solution for cell lysis and pre-modi cation of extracted DNA in the rst reservoir of 9 reservoirs near the outlet of a reagent reservoir channel (green-coloured dye). Secondly, we separately placed 120 µl of PBS in fourth, fth and sixth reagent reservoirs (each 40 µl of PBS) for the washing step of the DNA extraction (blue-coloured dye). Thirdly, we inserted 30 µl of 10 mM sodium bicarbonate (pH 10.6 elution buffer) in the ninth reagent reservoir for the elution of bound DNA (red-coloured dye). Fourthly, 36.8 µl of RPA master mixes used in the test of non-instrumented RPA devices without adding templates (DNA and DI water) were separately inserted into three reaction chambers and 13.2 µl of DI water was only added to the uppermost reaction chamber as a negative control. Lastly, the reaction chambers were covered with an optical adhesive lm.
Bi-functional imidoesters of DMA reversibly-covalently bind amine groups on the functionalised surface and the sticky ends of the fragmented DNA. Therefore, we modi ed the glass surface with amine groups using APTES, a well-known silane-coupling agent and covered a binding channel by the glass. Brie y, the APTES treatment was performed as followed sequences; i) oxygen plasma was used to activate silanol groups on a slide glass using NT-2 plasma cleaning system (Anatech Ltd., CA), ii) the activated glass was immersed with 2% APTES solution in 95% ethanol for 2 hours, iii) the glass was washed with ethanol and DI water for 5 min in three times and dried with a nitrogen stream and iv) nally, curing of the surface was carried out in a convection oven (Memmert, Frankfurt, Germany) at 120°C for 15 min.

Pump validation: Gas producibility and its longlasting ability
As mentioned above, the working principle of a hydration reaction-based powerless actuator is formed gas. The cover lm from the inlet of a microchannel is detached. And water is injected into the container of the powder mixture. The inlet hole is again covered by attaching a cover lm to transport pre-inserted reagents to a reaction zone in the microchannel. The pre-inserted reagents with trapped air between reagents would be sequentially owed and passed away the reaction zone with speci c reactions corresponded to treated chemical or immobilised receptors. If needed, wastes and test samples can be separated by blocking or opening outlets with cover lms in this process.
With the xed composition of the powder, the ow rates of reagents can be changed by different amounts of powder or water. In hydration reaction, variations of powder amount are more effective in changing the ow rates than water amount. Therefore, we used different amounts of the powder and 100 µl of water for hydration reaction to demonstrate corresponding ow rates in a simple straight channel (Fig. 3(a)).
Test devices were fabricated by a CO 2 laser cutting/engraving machine (Universal laser systems, AZ, USA) using PMMA sheets. A test device consists of an upper cover layer engraved with a millimeter ruler and has three sets of inlets and outlets for three microchannels. This channel layer includes three microchannels designed with 1 x 1 x 25 mm 3 (width x height x length) of dimension and three reagent reservoirs with ellipse shapes for each channel. This lower layer includes an 8 mm diameter of circular cylinder-shaped container for powder mixture, a plat lower cover layer. While the assembling process, 20 µl of green coloured dyes were placed on reagent reservoirs before attaching the upper cover layer.
In order to test the effect of solute amount difference in hydration reaction, we separately put 1, 5 and 10 mg of the powder into inlets of three microchannels in a test device and put 30, 50 and 100 mg of the powder into another test device. Then, hydration reactions were started by injecting 100 µl of water into powder containers and covering inlet holes with cover lm and formed gases pushed in dyes through channels to outlets of microchannels. A digital camera recorded the ow motion. The velocity of uid was obtained using elapsed time for 1 mm movement from the recorded video. Flow rates of uid were calculated using the velocities and the cross-section area of microchannels. The ow rates based on the experiment shown in Fig. 3(a) were plotted according to the amounts of the powder used for hydration reactions shown in Fig. 3(b). From the graph, results showed that a more signi cant amount of the powder generates higher ow rates. In addition, the ow rates can be easily controlled with the proper amount of the powder depending on the purposes (mixing, binding or sorting) of the applications for a relatively short length of a microchannel required applications (less than 25 mm).
To use the gas-driven pump for multi-step reactions, ow rate uniformity and long-lasting ability need to be investigated. For the investigation, we designed and fabricated a 1 m length of the microchannel and chose 10 mg of the powder to assume that a lower amount of the powder has a relatively high uniformity of ow rates (Fig. 3(c)). In the same way, 10 mg of the powder was placed in the powder container, and it was activated by 100 µl of water after covering an inlet hole with a cover lm. The ow motion was recorded with 1 min of the time interval for 2 hours, and the velocity of uid was obtained via ow distance per min from the recorded video. Then, ow rates of uid were calculated and plotted with time, as shown in Fig. 3(d). In the gure, the measured ow rates show that the gas-forming process is maintained by hydration reaction between the tiny amount of the powder (10 mg) and small water drop (100 µl) with a narrow range of ow rates (4 ~ 8 µl/min) for over 2 hours except for 10 min from the initial reaction. In the particular application that needs to avoid rapidly changing region from 0 to 10 min, we can cover an inlet hole with a cover lm at 10 minutes after putting water into the powder container.

Valve validation: Operational error tolerance
Here we validate the valve design to: (1) allow tolerance for manual handling error and (2) reduce wastage of eluted DNA solution. In a long channel, the hydration reaction ( Fig. 4(a)) causes the solution to move to the junction (Fig. 4(b)). At the channel junction after the lm cover is removed (Fig. 4(c)) the ratio of solution wastage is 50% (due to equal uidic resistance) or less. In the event, when the operator erroneously removed the lm cover slightly later (when some solution has passed the junction), the ratio of solution ow toward the waster chamber would be lesser. Only 17% of solution ow towards the waste chamber after the lm cover was removed in the test. During lling the chamber (Fig. 4(d) to (e)), a ratio of solution wastage is measured to be 11%, lower than when owing towards the reaction chamber due to the larger dimension of the chamber. The total loss of solution during this period (from Fig. 4(c) to (e)) is 14%. It is possible to reduce the ratio of solution wastage by designing the channel towards the reaction chamber to be shorter but with a larger cross-sectional area (See Fig. 4 (f)). Also, when the reaction chamber is lled up, the remaining uids ow to waste due to pressure-equalizing ( Fig. 4(e), (f)). If enough distance is implemented between reaction and waste solutions, the reaction and waste solutions can be separated naturally.
To counter this loss, it is advisable to include more sodium bicarbonate than the reaction chamber size. Moreover, the design of the valve enables the eluted DNA solution to completely clear into the waste channel once the reaction chamber is lled and prevent over owing (Fig. 4(d) and (e)) due to the presence of uidic gravitational pressure and surface tension. Therefore, the lm valve design is a suitable low-cost valve by compromising some losses in the eluted DNA solution.
3.3 RPA device validation : The RPA of nucleic acids with Exothermic reaction-based recombinant polymerase ampli cation (RPA) device We developed the exothermic reaction-based recombinant polymerase ampli cation (RPA) device, and its performance was evaluated. In the exothermic reaction, paper width and powder amount are key factors to achieve the desired temperature and duration of the heat-up process. Accordingly, temperature variations in time were plotted with different amount powders (0.4 and 0.5 g) and different paper widths (3 and 5 mm), as shown in Fig. 5(b). We inserted a digital probe thermometer with a timer (Oneida, USA) into a reaction chamber during the heat-up process and read temperatures from the thermometer per minute for the temperature measurements. In the experiments, the temperature increment is started at 10 min after adding 500 µl of water into the water container of the RPA device and the maximum temperature and overtime of exothermic reaction are varied with different experimental conditions (pale green, pale blue and pale red lines). Based on the results, we realised that the maximum temperature and the reaction over time could be controlled by the width of lter paper (i.e. ow rate of water) and the ratio of the total amount with the ow rate. Consequently, for the RPA method (37 ~ 42°C for 25 ~ 35 min), we chose 0.5 g of powder and 3 mm width of lter paper, which attains a temperature variation of ±0.5°C for more than 70 mins, comparable with Liu et al.'s 47 and Huang et al.'s 48 works (±3°C and ±2°C respectively).
To con rm the device's performance, RPA of nucleic acids was carried out with human genomic DNA using the reagents and protocols described in the TwistAmp Basic kit (TwistDX, UK). The optimised protocol is as follows; i) 29.5 µl of rehydration buffer, each 2.4 µl of 10 mM forward and reverse primers (forward:5'-GCA GCA TGT CAA GAT CAC AGA TTT TGG GCT and reverse:5'-CAT GTG TTA AAC AAT ACA GCT AGT GGG AAG GCA, 240 bp), 10.7 µl of distilled (DI) water and 2.5 µl of 50 ng/µl human genomic DNA for a test sample or 2.5 µl of nuclease-free water were mixed in 200 ml tubes, and the mixtures were vortexed. Two of 47.5 µl reaction mixtures were mixed with dried enzyme pellets which include dNTPs, adenosine59-triphosphate, phosphor-creatine, creatine kinase, and components of the proteins gp32 (single-stranded DNA binding protein), uvsX (recombinase), and the uvsY (recombinase load factor). To initiate the reactions, 2.5 µl of magnesium acetate (MgAc) was added to each tube. Each 50 µl of the nal reaction mixture was separately put into reaction chambers. Finally, reaction chambers were covered with cover lms, and reaction mixtures were incubated for 40 min after adding 500 µl of water to a water container (Fig. 5(c)).
The RPA amplicons were visualised by electrophoresis technique using 1% agarose gel with 1.5 µl of ethidium bromide (EtBr). Fig. 5(d) shows the electropherogram result of stained RPA amplicons with EtBr onto agarose matrix according to different sized molecules; three vertical lines indicate DNA ladder (M), ampli ed test sample (T) and no DNA template (N). For T and N lines, band intensities in the position of the red box directly represent the quantity of EGFR genes in RPA amplicons. Therefore, through the dark band in the T line, unlike the N line, we could realise that the RPA of nucleic acids is successfully performed with our RPA device.

Performance of Disposable DNA Analysis Chip with a Powerless Actuator : DNA analysis
The performance of the developed disposable DNA analysis chip with a powerless actuator was validated in this section. Fig. 6 represents the DNA analysis process in the whole system using coloured dyes. At the beginning of the process, urine sample was applied as followed order (a); i) an optical adhesive lm was detached from the holes above the powder container and the rst reagent reservoir, ii) 10 µl of the test sample was introduced to the rst reagent reservoir using 10 µl of a disposable pipette, iii) 100 µl of DI water was added to the powder container using 100 µl of a disposable pipette and iv) the holes were again covered with an optical adhesive lm at 10 min after injecting DI water. As a result, hydrogen gas was formed by the hydration reaction, and the gas started to push reagents in sequence from the mixture of urine sample, lysis buffer and DMA (green-coloured dye) to elution buffer (redcoloured dye) (Fig. 6(b)). In the spiral channels, the cell lysis and binding of DMA with extracted DNA simultaneously occurred. While the mixture was passed through a long meandering channel, the DNA/DMA complexes were captured by the amine group of APTES treated slide glass. According to the sequential ow, PBS (blue-coloured dye) was secondly owed through the long meandering channel to wash away non-speci c and unbound molecules. In a microchannel with multi-outlets, the ow directions of uids can be easily controlled by adjusting hydrodynamic resistances (R = 12μl /wh 3 ) among microchannels. In this work, to separately guide waste uids and extracted DNA uids, we designed two microchannels with different lengths to make the hydrodynamic resistance difference.
In addition, by blocking reaction chambers (i.e. outlets of the relatively shorter channel), we made to ow two solutions (green and blue-coloured dyes) toward a waste container, as shown in Fig. 6 (b-iii). For the next step, to collect extracted DNA, we detached an optical adhesive lm from the reaction chambers when the waste uid was passed entirely through the meandering channel. An elution buffer ows to the reaction chambers due to the high hydrodynamic resistance and hydraulic head of the waste uid. The chambers have relatively lower pressure through a meandering channel with eluted DNA by breaking the covalent bonds of amine groups between DNA and APTES, as shown in Fig. 6(c). During the process, we observed that the eluted DNA uid was evenly split into two reaction chambers by the same pressure drop from a junction to outlets of the chambers (Fig. 6(c-ii ~ iv)). Fig. 6(d) shows the operation process of RPA in the system chip. To minimise thermal losses via air in microchannels, a short bridge-channel made by optical adhesive lms was detached from the reaction chambers ( Fig. 6(d-i)). And a slide glass with 22 × 22 mm 2 areas was again attached to the chambers with double-sided tape (Fig. 6(d-ii)). Finally, by adding 500 µl of water to a water container using a disposable pipette, the exothermic reaction of the powder was started (Fig. 6(d-iii)) and DNA was incubated with RPA reagents for 40 min to amplify extracted DNA (Fig. 6(d-iv)).

Conclusion
Various concepts of micro uidic-based POC diagnostic assays and the LOC technologies have been reviewed and re ned in this study. The development of a simple, rapid, and low-cost device for quantitative nucleic acid detection is crucial for point-of-care (POC) diagnostic; simpli cation of the platform's power source and external components are the key to success.
We have developed an innovative yet straightforward system based on a hydration reaction. The hydrogen gas is generated by the hydration reaction of powder with water and expanded to push in reagents through the reaction zone toward the microchannel outlet. A cover lm controls the gas ow by attaching and detaching onto/from the microchannel holes. Finally, we have optimised ow conditions for DNA total analysis and demonstrated an exothermic reaction-based non-instrumented RPA device to prove our concept study. The chip is a fully disposable device that can perform DNA ampli cation via self-powered heating to be adopted in the POC diagnostic process. Various ampli ed target DNA detection methods can be considered based on objectives and environments for DNA analysis validation. We believe that our system would provide compatibility with any detection methods and enhance convenience and portability. Figure 1 Schematic diagram of working principle for hydration reaction-based powerless actuator; i) applying water & attaching cover lm and ii) producing hydrogen gas by hydration reaction & pushing reagent and basic design concept non-instrumented gas-driven micro uidic system. The formation of hydrogen gas from the powerless actuator is located in a microchannel inlet by applying water and covering the inlet hole. Sequential injection of pre-inserted reagents (red-blue-green) with air spacers between reagents through reaction zone to the microchannel outlet. Occurring reactions of reagents in the reaction zone according to the injection sequence. water & powder container component. Fabrication of all components by cutting/engraving PMMA sheets and double-sided tapes using a CO 2 laser machine and assembly process of components performed with double-sided tapes. v) Cover lms, bridge channels made by cutting optical adhesive lms.

Figure 3
Images of; (a) three straight microchannels with 1 × 1 × 25 mm 3 volume for the ow rate measurement of formed gas by hydration reaction across the time measured and (c) along microchannel with 1 × 1 × 1000 mm 3 volume for the determination of ow rate uniformity and the duration of hydration reaction.   (a) Image of structure of the non-instrumented RPA device. Adding a test sample into the left reaction reservoir and a solution for negative control into the right reaction reservoir and attaching an optical adhesive lm onto reaction chambers. (b) A graph of temperature variation plot with time using different combinations of exothermic reactive powder amounts (0.5 g and 0.4 g) and paper strip widths (3 mm and 5 mm). Initiate exothermic reactive powder by wicking water to the powder container 5 min after applying water to a water reservoir. 0.5 g amount of the powder and 3 mm width of the paper strip met the required temperature and time for the RPA of DNA. (c) Image of the RPA device with decided condition captured during the RPA. (d) Image of cropped gel electropherogram result indicating DNA ladder (M), ampli ed test sample (T) and negative control (N); full-length gel electropherogram is presented in Figure S1.