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Lab-on-a-chip systems with on-site capabilities offer the potential for rapid and accurate diagnosis and are useful in resource-constrained settings where biomedical equipment and trained professionals are not available. However, creating a point-of-care testing system that simultaneously has all the necessary features for multi-functional dispensing, on-demand release, reliable performance, and long-term storage of reagents remains a major challenge. Here we describe a lever-actuated micro travel switch technology that can manipulate fluids in any direction, provide precise and proportional response to applied air pressure, and remain stable against sudden movements and vibrations. Based on the technology, we also describe development of a polymerase chain reaction system that integrates reagent introduction, mixing and reaction functions all in one process, which accomplishes “sample-in-answer-out” performance for all clinical nasal samples from 18 patients with Influenza and 18 individual controls, in good concordance of fluorescence intensity with standard polymerase chain reaction (Pearson coefficients > 0.9). Based on the technology, we also describe the development of a polymerase chain reaction system that integrates reagent introduction, mixing and reaction functions all in one process, which accomplishes “sample-in-answer-out” performance for all clinical nasal samples from 18 patients with Influenza and 18 individual controls, in good concordance of fluorescence intensity with standard polymerase chain reaction (Pearson coefficients > 0.9). Основываясь на этой технологии, мы также описываем разработку системы полимеразной цепной реакции, которая объединяет функции введения реагентов, смешивания и реакции в одном процессе, что обеспечивает выполнение «образец-в-ответ-выход» для всех клинических образцов из носа от 18 пациентов с Грипп и 18 отдельных контролей, в хорошем соответствии интенсивности флуоресценции со стандартной полимеразной цепной реакцией (коэффициенты Пирсона> 0,9). Based on this technology, we also describe the development of a polymerase chain reaction system that combines the functions of injecting, mixing, and reacting in a single process, enabling sample-in-response-out for all clinical nasal specimens from 18 influenza patients. and 18 individual controls, in good agreement with the standard polymerase chain reaction fluorescence intensity (Pearson’s coefficients > 0.9). Based on this technology, we also describe the development of a polymerase chain reaction system that integrates reagent injection, mixing, and reaction functions to analyze all clinical nasal specimens from 18 in-sample nasal patient specimens.Influenza and 18 individual controls, fluorescence intensity matched well with standard polymerase chain reaction (Pearson’s coefficient > 0.9). The proposed platform guarantees reliable automation of biomedical analysis and thus can accelerate the commercialization of a range of point-of-care testing devices.
Emerging human diseases, such as the 2020 COVID-19 pandemic that has claimed the lives of millions of people, pose a serious threat to global health and human civilization1. Early, rapid and accurate detection of diseases is critical to control the spread of the virus and improve treatment outcomes. A core diagnostic ecosystem based on centralized labs where test samples are sent to hospitals or diagnostic clinics and run by professionals is currently restricting access for nearly 5.8 billion people worldwide, especially those living in resource-constrained settings. where there is a lack of expensive biomedical equipment and qualified specialists. clinicians 2. Thus, there is an urgent need to develop an inexpensive and user-friendly lab-on-a-chip system with point-of-care testing (POCT) capability that can provide clinicians with timely diagnostic information to make informed diagnosis decisions. and treatment 3.
The World Health Organization (WHO) guidelines state that an ideal POCT should be affordable, user-friendly (easy to use with minimal training), accurate (avoid false negatives or false positives), fast and reliable (provide good repeatability properties), and deliverable ( capable of long-term storage and readily available to end users)4. To meet these requirements, POCT systems must provide the following features: versatile dosing to reduce manual intervention, on-demand release to scale reagent transport for accurate test results, and reliable performance to withstand environmental vibration. Currently, the most widely used POCT device is the lateral flow strip5,6 consisting of several layers of porous nitrocellulose membranes that push a very small amount of sample forward, reacting with pre-immobilized reagents by capillary force. Although they have the advantage of low cost, ease of use, and rapid results, flow strip-based POCT devices can only be used for biological tests (e.g., glucose tests7,8 and pregnancy tests9,10) without requiring multi-stage analyses. reactions (eg loading of multiple reagents, mixing, multiplexing). In addition, the driving forces that control fluid movement (i.e., capillary forces) do not provide good consistency, especially between batches, resulting in poor reproducibility11 and making lateral flow bands primarily useful for good detection12,13.
Expanded manufacturing capabilities at the micro- and nanoscale have created opportunities for the development of microfluidic POCT devices for quantitative measurements14,15,16,17. By adjusting the properties of the interface 18, 19 and the geometry of the channels 20, 21, 22, the capillary force and flow rate of these devices can be controlled. However, their reliability, especially for highly wetted liquids, remains unacceptable due to manufacturing inaccuracies, material defects, and sensitivity to environmental vibrations. In addition, since a capillary flow is created at the liquid-gas interface, no additional flow can be introduced, especially after filling the microfluidic channel with liquid. Therefore, for more complex detection, several steps of sample injection must be performed24,25.
Among microfluidic devices, centrifugal microfluidic devices are currently one of the best solutions for POCT26,27. Its drive mechanism is advantageous in that the driving force can be controlled by adjusting the rotational speed. However, the disadvantage is that the centrifugal force is always directed towards the outer edge of the device, making it difficult to implement the multi-step reactions required for more complex analyses. Although additional driving forces (e.g. capillaries 28, 29 and many others 30, 31, 32, 33, 34, 35) in addition to the centrifugal force are introduced for multifunctional dosing, unforeseen liquid transfer can still occur because these additional forces are generally orders of magnitude lower than the centrifugal force, making them only effective over small operating ranges or not available on demand with liquid release. Incorporating pneumatic manipulations into centrifugal microfluidics such as centrifugal kinetic methods 36, 37, 38, thermopneumatic methods 39 and active pneumatic methods 40 has proven to be an attractive alternative. With the counterfugodynamic approach, an additional cavity and connecting microchannels are integrated into the device for both external and internal action, although its pumping efficiency (in the range from 75% to 90%) is highly dependent on the number of pumping cycles and the viscosity of the liquid. In the thermopneumatic method, the latex membrane and fluid transfer chamber are specifically designed to seal or reopen the inlet when the trapped air volume is heated or cooled. However, the heating/cooling setup introduces slow response problems and limits its use in thermosensitive assays (eg, polymerase chain reaction (PCR) amplification). With an active pneumatic approach, on-demand release and inward movement are achieved through the simultaneous application of positive pressure and precisely matched rotational speeds by high-speed motors. There are other successful approaches using only pneumatic actuators (positive pressure 41, 42 or negative pressure 43) and normally closed valve designs. By successively applying pressure in the pneumatic chamber, the liquid is pumped forward peristaltically, and the normally closed valve prevents the backflow of liquid due to peristalsis, thus realizing complex liquid operations. However, there are currently only a limited number of microfluidic technologies that can perform complex liquid operations in a single POCT device, including multi-functional dispensing, on-demand release, reliable performance, long-term storage, handling of high-viscosity liquids, and cost-effective manufacturing. All at the same time. The lack of a multi-step functional operation may also be one of the reasons why only a few commercial POCT products such as Cepheid, Binx, Visby, Cobas Liat, and Rhonda have been successfully introduced on the open market to date.
In this paper, we propose a pneumatic microfluidic actuator based on green ring micro switch technology (FAST). FAST combines all the necessary properties at the same time for a wide range of reagents from microliters to milliliters. FAST consists of elastic membranes, levers and blocks. Without the application of air pressure, the membranes, levers and blocks can be tightly closed and the liquid inside can be stored for a long time. When appropriate pressure is applied and adjusted to the length of the lever, the diaphragm expands and pushes the lever to the open position, allowing fluid to pass through. This allows multifunctional metering of liquids in a cascade, simultaneous, sequential or selective manner.
We have developed a PCR system using FAST to generate response-in-sample results for the detection of influenza A and B viruses (IAV and IBV). We achieved a lower limit of detection (LOD) of 102 copies/mL, our multiplex assay showed specificity for IAV and IBV and allowed influenza virus pathotyping. The clinical testing results using the nasal swab sample from 18 patients and 18 healthy individuals show good concordance in fluorescence intensity with standard RT-PCR (Pearson coefficients > 0.9). The clinical testing results using the nasal swab sample from 18 patients and 18 healthy individuals show good concordance in fluorescence intensity with standard RT-PCR (Pearson coefficients > 0.9). Результаты клинических испытаний с использованием образца мазка из носа от 18 пациентов и 18 здоровых лиц показывают хорошее соответствие интенсивности флуоресценции стандартной ОТ-ПЦР (коэффициенты Пирсона > 0,9). The results of clinical trials using a nasal swab sample from 18 patients and 18 healthy individuals show good agreement between the fluorescence intensity of standard RT-PCR (Pearson’s coefficients > 0.9). 0.9）。。。。。。。。。。。。。。。。。。。。。。。。。 Результаты клинических испытаний с использованием образцов назальных мазков от 18 пациентов и 18 здоровых лиц показали хорошее соответствие между интенсивностью флуоресценции и стандартной ОТ-ПЦР (коэффициент Пирсона > 0,9). The results of clinical trials using nasal swab specimens from 18 patients and 18 healthy individuals showed good agreement between fluorescence intensity and standard RT-PCR (Pearson’s coefficient > 0.9). The estimated material cost of a FAST-POCT device is approximately US$1 (Supplementary Table 1) and can be further reduced by using large scale manufacturing methods (eg injection molding). In fact, FAST-based POCT devices have all the necessary features mandated by WHO and are compatible with new biochemical testing methods such as plasma thermal cycling44, amplification-free immunoassays45 and nanobody functionalization tests46 that are the backbone of POCT systems. possibility.
On fig. 1a shows the structure of the FAST-POCT platform, which consists of four liquid chambers: a pre-storage chamber, a mixing chamber, a reaction chamber, and a waste chamber. The key to fluid flow control is the FAST design (consisting of elastic membranes, levers and blocks) located in the pre-storage chamber and mixing chamber. As a pneumatically actuated method, the FAST design provides precise fluid flow control, including closed/open switching, versatile dosing, on-demand fluid release, reliable operation (e.g., insensitivity to environmental vibration), and long-term storage. The FAST-POCT platform consists of four layers: a backing layer, an elastic film layer, a plastic film layer, and a cover layer, as shown in an enlarged view in Fig. 1b (also shown in detail in Supplementary Figures S1 and S2). All channels and fluid transport chambers (such as pre-storage and reaction chambers) are embedded in PLA (polylactic acid) substrates ranging from 0.2 mm (thinnest part) to 5 mm thick. The elastic film material is a 300 µm thick PDMS that easily expands when air pressure is applied due to its “thin thickness” and low modulus of elasticity (about 2.25 MPa47). The polyethylene film layer is made of polyethylene terephthalate (PET) with a thickness of 100 µm to protect the elastic film from excessive deformation due to air pressure. Corresponding to the chambers, the substrate layer has levers connected to the cover layer (made of PLA) by hinges to control the flow of liquid. The elastic film was glued to the backing layer using double-sided adhesive tape (ARseal 90880) and covered with a plastic film. Three layers were assembled on a substrate using a T-clip design in the cover layer. The T-clamp has a gap between two legs. When the clip was inserted into the groove, the two legs bent slightly, then returned to their original state and tightly bound the lid and backing as they passed through the groove (Supplementary Fig. S1). The four layers are then assembled using connectors.
Schematic diagram of the platform illustrating the various functional compartments and features of FAST. b Enlarged diagram of the FAST-POCT platform. c Photo of the platform next to a US quarter dollar coin.
The working mechanism of the FAST-POCT platform is shown in Figure 2. The key components are the blocks on the base layer and the hinges on the cover layer, which results in an interference design when the four layers are assembled using a T-shape. When no air pressure is applied (fig. 2a), the interference fit causes the hinge to bend and deform, and a sealing force is applied through the lever to press the elastic film against the block, and the liquid in the seal cavity is defined as a sealed state. It should be noted that in this state, the lever is bent outward, as shown in the side view in Fig. 2a. When air is supplied (Fig. 2b), the elastic membrane expands outward towards the cover and pushes the lever up, thus opening a gap between the lever and the block for fluid to flow into the next chamber, which is defined as an open state. After the release of air pressure, the lever can return to its original position and remain tight due to the elasticity of the hinge. Videos of the lever movements are presented in the supplementary movie S1.
A. Schematic diagram and photographs when closed. In the absence of pressure, the lever presses the membrane against the block, and the liquid is sealed. b In good condition. When pressure is applied, the membrane expands and pushes the lever up, so the channel opens and fluid can flow. c Determine the characteristic size of the critical pressure. Characteristic dimensions include the length of the lever (L), the distance between the slider and the hinge (l) and the thickness of the lever protrusion (t). Fs is the compaction force at throttle point B. q is the uniformly distributed load on the lever. Tx* represents the torque developed by the hinged lever. Critical pressure is the pressure required to raise the lever and make the fluid flow. d Theoretical and experimental results of the relationship between critical pressure and element size. n = 6 independent experiments were performed and data are shown as ± standard deviation. Raw data are presented as raw data files.
An analytical model based on the beam theory has been developed to analyze the dependence of the critical pressure Pc at which the gap opens on the geometric parameters (for example, L is the length of the lever, l is the distance between the block and the hinge, S is the lever The contact area with the liquid t is the thickness of the lever protrusion , as shown in Fig. 2c). As detailed in the Supplementary Notes and Supplementary Figure S3, the gap opens when \({P}_{c}\ge \frac{2{F}_{s}l}{SL}\), where Fs is the torque \ ({T}_{x}^{\ast}(={F}_{s}l)\) to eliminate the forces associated with an interference fit and cause the hinge to bend. The experimental response and the analytical model show good agreement (Fig. 2d), showing that the critical pressure Pc increases with increasing t/l and decreasing L, which is easily explained by the classical beam model, i.e. the torque increases with t /Lift . Thus, our theoretical analysis clearly shows that the critical pressure can be effectively controlled by adjusting the lever length L and the t/l ratio, which provides an important basis for the design of the FAST-POCT platform.
The FAST-POCT platform provides multifunctional dispensing (shown in Figure 3a with inset and experiment), which is the most important feature of successful POCT, where fluids can flow in any direction and in any order (cascade, simultaneous, sequential) or selective multichannel dispensing . – dosing function. On fig. 3a(i) shows a cascaded dosing mode in which two or more chambers are cascaded using blocks to separate the various reactants and a lever to control the open and closed states. When pressure is applied, the liquid flows from the upper to the lower chamber in a cascade manner. It should be noted that the cascade chambers can be filled with wet chemicals or dry chemicals such as lyophilized powders. In the experiment in Fig. 3a(i), the red ink from the upper chamber flows along with the blue dye powder (copper sulfate) into the second chamber and turns dark blue when it reaches the lower chamber. It also shows the control pressure for the fluid being pumped. Similarly, when one lever is connected to two chambers, it becomes the simultaneous injection mode, as shown in fig. 3a(ii), in which the liquid can be uniformly distributed over two or more chambers when pressure is applied. Since the critical pressure depends on the length of the lever, the length of the lever can be adjusted to achieve a sequential injection pattern as shown in fig. 3a(iii). A long lever (with critical pressure Pc_long) was connected to chamber B and a short lever (with critical pressure Pc_short > Pc_long) was connected to chamber A. As pressure P1 (Pc_long < P1 < Pc_short) was applied, only the liquid in red can flow to chamber B and when the pressure was increased to P2 (> Pc_short), the blue liquid can flow to chamber A. This sequential injection mode applies to different liquids transferring to their related chambers in sequence, which is critical for a successful POCT device. A long lever (with critical pressure Pc_long) was connected to chamber B and a short lever (with critical pressure Pc_short > Pc_long) was connected to chamber A. As pressure P1 (Pc_long < P1 < Pc_short) was applied, only the liquid in red can flow to chamber B and when the pressure was increased to P2 (> Pc_short), the blue liquid can flow to chamber A. This sequential injection mode applies to different liquids transferring to their related chambers in sequence, which is critical for a successful POCT device. Длинный рычаг (с критическим давлением Pc_long) был соединен с камерой B, а короткий рычаг (с критическим давлением Pc_short > Pc_long) был соединен с камерой A. При приложении давления P1 (Pc_long < P1 < Pc_short) только жидкость, выделенная красным может течь в камеру B, и когда давление было увеличено до P2 (> Pc_short), синяя жидкость может течь в камеру A. Этот режим последовательного впрыска применяется к различным жидкостям, последовательно перемещаемым в соответствующие камеры, что имеет решающее значение для успешной POCT. A long lever (with critical pressure Pc_long) was connected to chamber B, and a short lever (with critical pressure Pc_short > Pc_long) was connected to chamber A. When pressure P1 (Pc_long < P1 < Pc_short) is applied, only the liquid highlighted in red can flow into chamber B, and when the pressure has been increased to P2 (> Pc_short), the blue liquid can flow into chamber A. This sequential injection mode is applied to different fluids sequentially transferred to the respective chambers, which is critical for successful POCT. device. Длинный рычаг (критическое давление Pc_long) соединен с камерой B, а короткий рычаг (критическое давление Pc_short > Pc_long) соединен с камерой A. The long arm (critical pressure Pc_long) is connected to chamber B and the short arm (critical pressure Pc_short > Pc_long) is connected to chamber A.При приложении давления P1 (Pc_long < P1 < Pc_short) в камеру B может поступать только красная жидкость, а при увеличении давления до P2 (> Pc_short) в камеру A может поступать синяя жидкость. When pressure P1 (Pc_long < P1 < Pc_short) is applied, only red liquid can enter chamber B, and when pressure is increased to P2 (> Pc_short), blue liquid can enter chamber A. This sequential injection mode is suitable for sequential transfer of various fluids into the respective chambers, which is critical for the successful operation of the POCT device. Figure 3a(iv) demonstrates the selective injection mode, where the main chamber had a short (with critical pressure Pc_short) and a long lever (with critical pressure Pc_long < Pc_short) that were connected to chamber A and chamber B, respectively, in addition to another air channel connected to chamber B. To transfer the liquid to chamber A first, pressure P1 (Pc_long < P1 < Pc_short) and P2 (P2 > P1) with P1 + P2 > Pc_short were applied to the device at the same time. Figure 3a(iv) demonstrates the selective injection mode, where the main chamber had a short (with critical pressure Pc_short) and a long lever (with critical pressure Pc_long < Pc_short) that were connected to chamber A and chamber B, respectively, in addition to another air channel connected to chamber B. To transfer the liquid to chamber A first, pressure P1 (Pc_long < P1 < Pc_short) and P2 (P2 > P1) with P1 + P2 > Pc_short were applied to the device at the same time. On fig. 3а(iv) показан режим селективного впрыска, при котором основная камера имела короткий (с критическим давлением Pc_short) и длинный рычаг (с критическим давлением Pc_long < Pc_short), которые дополнительно соединялись с камерой A и камерой B соответственно. 3a(iv) shows the selective injection mode, in which the main chamber had a short (with critical pressure Pc_short) and a long lever (with critical pressure Pc_long < Pc_short), which were additionally connected to chamber A and chamber B, respectively. к другому воздушному каналу, соединенному с камерой B. Чтобы сначала передать жидкость в камеру A, к устройству одновременно прикладывали давление P1 (Pc_long < P1 < Pc_short) и P2 (P2 > P1), где P1 + P2 > Pc_short. to another air channel connected to chamber B. To first transfer fluid to chamber A, pressures P1 (Pc_long < P1 < Pc_short) and P2 (P2 > P1) were simultaneously applied to the device, where P1 + P2 > Pc_short. 3а(iv) показан режим селективного впрыска, когда основная камера имеет короткий стержень (с критическим давлением Pc_short) и длинный стержень (с критическим давлением Pc_long < Pc_short), соединенные с камерой A и камерой B соответственно, и в дополнение к другому воздушному каналу, подключенному к комнате B. 3a(iv) shows the selective injection mode when the main chamber has a short stem (critical pressure Pc_short) and a long stem (critical pressure Pc_long < Pc_short) connected to chamber A and chamber B respectively, and in addition to another air passage, connected to room B. Thus, P2 prevents liquid from entering chamber B; meanwhile, the total pressure P1 + P2 exceeded the critical pressure to activate the shorter lever connected to chamber A to allow the liquid flow to chamber A. Then, when chamber B was required to be filled, we only need to apply P1 (Pc_long < P1 < Pc_short) in the main chamber to activate the long lever and allow the liquid to flow to chamber B. It can be clearly observed from time t = 3 s to 9 s that the liquid in chamber A remained constant while it increased in chamber B when pressure P1 was applied. meanwhile, the total pressure P1 + P2 exceeded the critical pressure to activate the shorter lever connected to chamber A to allow the liquid flow to chamber A. Then, when chamber B was required to be filled, we only need to apply P1 (Pc_long < P1 < Pc_short) in the main chamber to activate the long lever and allow the liquid to flow to chamber B. It can be clearly observed from time t = 3 s to 9 s that the liquid in chamber A remained constant while it increased in chamber B when pressure P1 was applied. Между тем, общее давление P1 + P2 превысило критическое давление, чтобы активировать более короткий рычаг, соединенный с камерой A, чтобы позволить жидкости течь в камеру A. Затем, когда требуется заполнить камеру B, нам нужно только применить P1 (Pc_long < P1 < Pc_short) в основной камере, чтобы активировать длинный рычаг и дать жидкости течь в камеру B. Можно ясно наблюдать, что в период с t = 3 с до 9 с жидкость в камере A оставалась постоянной, в то время как в камере она увеличивалась. Meanwhile, the total pressure P1 + P2 has exceeded the critical pressure to activate a shorter lever connected to chamber A to allow liquid to flow into chamber A. Then when chamber B needs to be filled, we only need to apply P1 (Pc_long < P1 < Pc_short ) in the main chamber to activate the long lever and let the liquid flow into chamber B. It can be clearly observed that between t = 3 s and 9 s the liquid in chamber A remained constant, while in chamber it increased. B when pressure P1 is applied. At the same time, the total pressure P1 + P2 exceeds the critical pressure, actuating the shorter lever connecting chamber A, allowing fluid to flow into chamber A.When it’s time to fill chamber A, we simply apply P1 in the main chamber and P2 in the secondary chamber. In this way, the flow behavior can be selectively switched between cameras A and B. The flow behavior of the four multi-functional distribution modes can be found in the supplementary movie S2.
a Illustration of multifunctional assignment, i.e. (i) cascading, (ii) simultaneous, (iii) sequential, and (iv) selective assignment. The curves represent the workflow and parameters of these four distribution modes. b Results of long-term storage tests in deionized water and ethanol. n = 5 independent experiments were performed and data are shown as ± sd c. Stability test demonstrations when the FAST device and the capillary valve (CV) device were in (i) static and (ii) vibrating states. (iii) Volume vs. time for FAST and CV devices at various angular frequencies. d Publication of test results on demand for (i) FAST device and (ii) CV device. (iii) Relationship between volume and time for FAST and CV devices using intermittent pressure mode. All scale bars, 1 cm. Raw data are provided as raw data files.
Long-term storage of reagents is another important feature of a successful POCT device that will allow untrained personnel to handle multiple reagents. While many technologies have shown their potential for long-term storage (eg, 35 microdispensers, 48 blister packs, and 49 stick packs), a dedicated receiving compartment is required to accommodate the package, which increases cost and complexity; furthermore, these storage mechanisms do not allow for on-demand dispensing and result in wastage of reagents due to leftovers in the packaging. Long-term storage capability was verified by conducting an accelerated life test using CNC-machined PMMA material due to its slight roughness and resistance to gas permeation (Supplementary Figure S5). The test apparatus was filled with deionized water (deionized water) and 70% ethanol (simulating volatile reagents) at 65°C for 9 days. Both deionized water and ethanol were stored using aluminum foil to block access from above. The Arrhenius equation and penetration activation energy reported in the literature50,51 were used to calculate the real-time equivalent. On fig. 3b shows the average weight loss results for 5 samples stored at 65°C for 9 days, equivalent to 0.30% for deionized water and 0.72% for 70% ethanol over 2 years at 23°C.
On fig. 3c shows the vibration test. Since the capillary valve (CV) is the most popular fluid handling method among existing POCT28,29 devices, a CV device 300 µm wide and 200 µm deep was used for comparison. It can be seen that when both devices remain stationary, the fluid in the FAST-POCT platform seals and the fluid in the CV device locks up due to the sudden expansion of the channel, which reduces capillary forces. However, as the orbital vibrator’s angular frequency increases, the fluid in the FAST-POCT platform remains sealed, but the fluid in the CV device flows into the lower chamber (see also Supplementary Movie S3). This suggests that the deformable hinges of the FAST-POCT platform can apply a strong mechanical force to the module to tightly close the liquid in the chamber. However, in CV devices, liquid is retained due to the balance between the solid, air, and liquid phases, creating instability, and vibrations can upset the balance and cause unexpected flow behavior. The advantage of the FAST-POCT platform is that it provides reliable functionality and avoids failures in the presence of vibrations that usually occur during delivery and operation.
Another important feature of the FAST-POCT platform is its on-demand release, which is a key requirement for quantitative analysis. On fig. 3d compares the on-demand release of the FAST-POCT platform and the CV device. From fig. 3d(iii) we see that the FAST device responds quickly to the pressure signal. When pressure was applied to the FAST-POCT platform, the fluid flowed, when the pressure was released, the flow immediately stopped (Fig. 3d(i)). This action can be explained by the quick elastic return of the hinge, which presses the lever back against the block, closing the chamber. However, fluid continued to flow in the CV device, eventually resulting in an unexpected fluid volume of approximately 100 µl after pressure was released (Figure 3d(ii) and Supplementary Movie S4). This can be explained by the disappearance of the capillary pinning effect upon complete wetting of the CV after the first injection.
The ability to handle liquids of varying wettability and viscosity in the same device remains a challenge for POCT applications. Poor wettability can lead to leaks or other unexpected flow behavior in the channels, and ancillary equipment such as vortex mixers, centrifuges and filters are often required to prepare highly viscous liquids 52 . We tested the relationship between critical pressure and fluid properties (with a wide range of wettability and viscosity). The results are shown in Table 1 and Video S5. It can be seen that liquids of different wettability and viscosity can be sealed in the chamber, and when pressure is applied, even liquids with a viscosity of up to 5500 cP can be transferred to the adjacent chamber, making it possible to detect samples with high viscosity (i.e. sputum, a very viscous sample used for the diagnosis of respiratory diseases).
By combining the above multifunctional dispensing devices, a wide range of FAST-based POCT devices can be developed. An example is shown in Figure 1. The plant contains a pre-storage chamber, a mixing chamber, a reaction chamber and a waste chamber. Reagents may be stored in the pre-storage chamber for extended periods of time and then discharged into the mixing chamber. With the right pressure, the mixed reactants can be selectively transferred to a waste chamber or a reaction chamber.
Because PCR detection is the gold standard for detecting pathogens such as H1N1 and COVID-19 and involves multiple reaction steps, we used the FAST-POCT platform for PCR detection as an application. On fig. 4 shows the PCR testing process using the FAST-POCT platform. First, the eluting reagent, magnetic microbead reagent, wash solution A, and wash solution W were pipetted into pre-storage chambers E, M, W1 and W2, respectively. The stages of RNA adsorption are shown in fig. 4a and are as follows: (1) when pressure P1 (=0.26 bar) is applied, the sample moves into chamber M and is discharged into the mixing chamber. (2) Air pressure P2 (= 0.12 bar) is supplied through port A connected to the bottom of the mixing chamber. Although a number of mixing methods have shown their potential in mixing liquids on POCT platforms (eg serpentine mixing 53, random mixing 54 and batch mixing 55), their mixing efficiency and effectiveness are still not satisfactory. It adopts the bubble mixing method, in which air is introduced into the bottom of the mixing chamber to create bubbles in the liquid, after which the powerful vortex can achieve complete mixing within seconds. Bubble mixing experiments were carried out and the results are presented in Supplementary Figure S6. It can be seen that when a pressure of 0.10 bar is applied, complete mixing takes about 8 seconds. By increasing the pressure to 0.20 bar, complete mixing is achieved in about 2 seconds. Methods for calculating mixing efficiency are presented in the Methods section. (3) Use a rubidium magnet to extract the beads, then pressurize P3 (= 0.17 bar) through port P to move the reagents into the waste chamber. On fig. 4b,c shows the washing steps to remove impurities from the sample as follows: (1) The washing solution A from the chamber W1 is discharged into the pressure mixing chamber P1. (2) Then do the bubble mixing process. (3) The washing solution A is transferred to the waste liquid chamber, and the microbeads in the mixing chamber are pulled out by the magnet. Washing W (Fig. 4c) was similar to washing A (Fig. 4b). It should be noted that each washing step A and W was performed twice. Figure 4d shows the elution steps to elute the RNA from the beads; the elution and mixing introduction steps are the same as the RNA adsorption and washing steps described above. As the elution reagents are transferred into the PCR reaction chamber under pressures P3 and P4 (=0.23 bar), the critical pressure is reached to seal the arm of the PCR reaction chamber. Similarly, the P4 pressure also helps to seal the passage to the waste chamber. Thus, all elution reagents were evenly distributed among the four PCR reaction chambers to initiate the multiplex PCR reactions. The above procedure is presented in Supplementary Movie S6.
In the RNA adsorption step, the sample is introduced into the inlet M and injected into the mixing chamber along with the previously stored bead solution. After mixing and removing the granules, the reagents are distributed into the waste chamber. b and c wash steps, introduce various pre-stored wash reagents into the mixing chamber, and after mixing and removing the beads, transfer the reagents to the waste liquid chamber. d Elution step: After introducing the elution reagents, mixing and bead extraction, the reagents are transferred to the PCR reaction chamber. The curves show the workflow and related parameters of the various stages. Pressure is the pressure exerted through the individual chambers. Volume is the volume of liquid in the mixing chamber. All scale bars are 1 cm. Raw data are provided as raw data files.
A PCR testing procedure was performed and Supplementary Figure S7 presents thermal profiles including 20 minutes of reverse transcription time and 60 minutes of thermal cycling time (95 and 60 °C), with one thermal cycle being 90 s (Supplementary Movie S7). . FAST-POCT requires less time to complete one thermal cycle (90 seconds) than conventional RT-PCR (180 seconds for one thermal cycle). This can be explained by the high surface area to volume ratio and the low thermal inertia of the micro-PCR reaction chamber. The chamber surface is 96.6 mm2 and the chamber volume is 25 mm3, making the surface to volume ratio approximately 3.86. As seen in Supplementary Figure S10, the PCR test area of our platform has a groove on the back panel, making the bottom of the PCR chamber 200 µm thick. A thermally conductive elastic pad is attached to the heating surface of the temperature controller, ensuring tight contact with the back of the test box. This reduces the thermal inertia of the platform and improves the heating/cooling efficiency. During thermal cycling, the paraffin embedded in the platform melts and flows into the PCR reaction chamber, acting as a sealant to prevent reagent evaporation and environmental contamination (see Supplementary Movie S8).
All PCR detection processes described above were fully automated using a custom made FAST-POCT instrument, consisting of a programmed pressure control unit, a magnetic extraction unit, a temperature control unit, and a fluorescent signal capture and processing unit. Of note, we used the FAST-POCT platform for RNA isolation and then used the extracted RNA samples for PCR reactions using the FAST-POCT system and desktop PCR system for comparison. The results were almost the same as shown in Supplementary Figure S8. The operator performs a simple task: introduces the sample into the M-chamber and inserts the platform into the instrument. Quantitative test results are available in about 82 minutes. Detailed information about FAST-POCT tools can be found in the supplementary figure. C9, C10 and C11.
Influenza caused by influenza A (IAV), B (IBV), C (ICV) and D (IDV) viruses is a common global phenomenon. Of these, IAV and IBV are responsible for the most severe cases and seasonal epidemics, infecting 5-15% of the world’s population, causing 3-5 million severe cases and causing 290,000-650,000 deaths annually. Respiratory diseases56,57. Early diagnosis of IAV and IB is essential to reduce morbidity and associated economic burden. Among available diagnostic techniques, the reverse transcriptase polymerase chain reaction (RT-PCR) is considered the most sensitive, specific, and accurate (>99%)58,59. Among available diagnostic techniques, the reverse transcriptase polymerase chain reaction (RT-PCR) is considered the most sensitive, specific, and accurate (>99%)58,59. Среди доступных диагностических методов полимеразная цепная реакция с обратной транскриптазой (ОТ-ПЦР) считается наиболее чувствительной, специфичной и точной (> 99%)58,59. Among the available diagnostic methods, reverse transcriptase polymerase chain reaction (RT-PCR) is considered the most sensitive, specific and accurate (> 99%)58,59. Из доступных диагностических методов полимеразная цепная реакция с обратной транскриптазой (ОТ-ПЦР) считается наиболее чувствительной, специфичной и точной (>99%)58,59. Of the available diagnostic methods, reverse transcriptase polymerase chain reaction (RT-PCR) is considered the most sensitive, specific and accurate (>99%)58,59. However, traditional RT-PCR methods require repeated pipetting, mixing, dispensing and transfer of fluid, limiting their use by professionals in resource-limited settings. Here, the FAST-POCT platform was used for PCR detection of IAV and IBV, respectively, to obtain their lower limit of detection (LOD). In addition, IAV and IBV have been multiplexed to discriminate between different pathotypes across species, providing a promising platform for genetic analysis and the ability to accurately treat the disease.
On fig. 5a shows the results of HAV PCR testing using 150 µl of purified viral RNA as a sample. On fig. 5a(i) shows that at an HAV concentration of 106 copies/ml, the fluorescence intensity (ΔRn) can reach 0.830, and when the concentration is reduced to 102 copies/ml, ΔRn can still reach 0.365, which corresponds to higher than that of the empty negative control group ( 0.002), about 100 times higher. For quantification based on six independent experiments, a linear calibration curve was generated between log concentration and cycle threshold (Ct) of IAV (Fig. 5a(ii)), R2 = 0.993, ranging from 102-106 copies/mL. the results are in good agreement with conventional RT-PCR methods. On fig. 5a(iii) shows fluorescent images of test results after 40 cycles of the FAST-POCT platform. We found that the FAST-POCT platform can detect HAV as low as 102 copies/mL. However, the traditional method does not have a Ct value at 102 copies/mL, making it an LOD of about 103 copies/mL. We hypothesized that this may be due to the high efficiency of bubble mixing. PCR test experiments were performed on purified IAV RNA to evaluate various mixing methods, including shake mixing (same mixing method as in conventional RT-PCR operation), vial mixing (this method, 3 s at 0.12 bar) and no mixing as a control group. . The results can be found in Supplementary Figure S12. It can be seen that at a higher RNA concentration (106 copies/mL), the Ct values of the different mixing methods are almost the same as for bubble mixing. When the RNA concentration dropped to 102 copies/mL, the shake mix and controls had no Ct values, while the bubble mix method still gave a Ct value of 36.9, which was below the Ct threshold of 38. The results show a dominant mixing characteristic vesicles, which has also been demonstrated in other literature, which may also explain why the sensitivity of the FAST-POCT platform is slightly higher than conventional RT-PCR. On fig. 5b shows the results of PCR analysis of purified IBV RNA samples ranging from 101 to 106 copies/ml. The results were similar to the IAV test, achieving an R2 = 0.994 and a LOD of 102 copies/mL.
a PCR analysis of influenza A virus (IAV) with IAV concentrations ranging from 106 to 101 copies/mL using TE buffer as a negative control (NC). (i) Real time fluorescence curve. (ii) Linear calibration curve between logarithmic IAV RNA concentration and cycle threshold (Ct) for FAST and conventional testing methods. (iii) IAV FAST-POCT fluorescent image after 40 cycles. b, PCR detection of influenza B virus (IBV) with (i) real-time fluorescence spectrum. (ii) Linear calibration curve and (iii) FAST-POCT IBV fluorescence image after 40 cycles. The lower limit of detection (LOD) for IAV and IBV using the FAST-POCT platform was 102 copies/mL, which is lower than conventional methods (103 copies/mL). c Multiplex test results for IAV and IBV. GAPDH was used as a positive control and TE buffer was used as a negative control to prevent possible contamination and background amplification. Four different sample types can be distinguished: (1) GAPDH-only negative samples (“IAV-/IBV-”); (2) IAV infection (“IAV+/IBV-”) with IAV and GAPDH; (3) IBV infection (“IAV-/IBV+”) with IBV and GAPDH; (4) IAV/IBV infection (“IAV+/IBV+”) with IAV, IBV and GAPDH. The dotted line represents the threshold line. n = 6 biologically independent experiments were performed, data are shown as ± standard deviation. Raw data are presented as raw data files.
On fig. 5c shows the results of the multiplexing test for IAV/IBV. Here, virus lysate was used as a sample solution in place of purified RNA, and four primers for IAV, IBV, GAPDH (positive control) and TE buffer (negative control) were added to four different reaction chambers of the FAST-POCT platform. Positive and negative controls are used here to prevent possible contamination and background enhancement. The tests were divided into four groups: (1) GAPDH-negative samples (“IAV-/IBV-”); (2) IAV-infected (“IAV+/IBV-”) versus IAV and GAPDH; (3) IBV-. infected (“IAV-”) -/IBV+”) IBV and GAPDH; (4) IAV/IBV (“IAV+/IBV+”) infection with IAV, IBV and GAPDH. On fig. 5c shows that when negative samples were applied, the fluorescence intensity ΔRn of the positive control chamber was 0.860, and the ΔRn of IAV and IBV was similar to the negative control (0.002). For the IAV+/IBV-, IAV-/IBV+ and IAV+/IBV+ groups, the IAV/GAPDH, IBV/GAPDH and IAV/IBV/GAPDH cameras showed significant fluorescence intensity, respectively, while the other cameras even showed fluorescence intensity at a background level of 40 after thermal cycling. From the tests above, the FAST-POCT platform showed outstanding specificity and allowed us to simultaneously pathotype different influenza viruses.
To validate the clinical applicability of FAST-POCT, we tested 36 clinical specimens (nose swab specimens) from IB patients (n=18) and non-IB controls (n=18) (Figure 6a). Patient information is presented in Supplementary Table 3. IB infection status was independently confirmed and the study protocol was approved by Zhejiang University First Affiliated Hospital (Hangzhou, Zhejiang). Each sample of patients was divided into two categories. One was processed using FAST-POCT and the other was processed using a desktop PCR system (SLAN-96P, China). Both assays use the same purification and detection kits. On fig. 6b shows the results of FAST-POCT and conventional reverse transcription PCR (RT-PCR). We compared the fluorescence intensity (FAST-POCT) with -log2(Ct), where Ct is the cycle threshold for conventional RT-PCR. There was good agreement between the two methods. FAST-POCT and RT-PCR showed a strong positive correlation with a Pearson’s ratio (r) value of 0.90 (Figure 6b). We then assessed the diagnostic accuracy of FAST-POCT. Fluorescence intensity (FL) distributions for positive and negative samples were provided as an independent analytical measure (Fig. 6c). FL values were significantly higher in IB patients than in controls (****P = 3.31 × 10-19; two-tailed t-test) (Fig. 6d). Next, IBV receiver operating characteristics (ROC) curves were plotted. We found that the diagnostic accuracy was very good, with an area under the curve of 1 (Fig. 6e). Please note that due to mandatory mask ordering in China due to COVID-19 as of 2020, we have not identified patients with IBD, so all positive clinical specimens (i.e., nasal swab specimens) were for IBV only.
Clinical study design. A total of 36 samples, including 18 patient samples and 18 non-influenza controls, were analyzed using the FAST-POCT platform and conventional RT-PCR. b Assess the analytical consistency between FAST-POCT PCR and conventional RT-PCR. The results were positively correlated (Pearson r = 0.90). c Fluorescence intensity levels in 18 IB patients and 18 controls. d In IB patients (+), FL values were significantly higher than in the control group (-) (****P = 3.31 × 10-19; two-tailed t-test; n = 36). For each square plot, the black marker in the center represents the median, and the bottom and top lines of the box represent the 25th and 75th percentiles, respectively. The whiskers extend to the minimum and maximum data points, which are not considered outliers. e ROC curve. The dotted line d represents the threshold value estimated from the ROC analysis. The AUC for IBV is 1. Raw data are provided as raw data files.
In this article, we present FAST, which has the characteristics required for an ideal POCT. The advantages of our technology include: (1) Versatile dosing (cascade, simultaneous, sequential and selective), release on demand (rapid and proportional release of applied pressure) and reliable operation (vibration at 150 degrees) (2) long-term storage (2 years of accelerated testing, weight loss about 0.3%); (3) the ability to work with liquids with a wide range of wettability and viscosity (viscosity up to 5500 cP); (4) Economical (Estimated material cost of the FAST-POCT PCR device is approximately US$1). By combining multifunctional dispensers, an integrated FAST-POCT platform for PCR detection of influenza A and B viruses was demonstrated and applied. FAST-POCT only takes 82 minutes. The clinical tests with 36 nasal swab samples showed good concordance in fluorescence intensity with standard RT-PCR (Pearson coefficients > 0.9). The clinical tests with 36 nasal swab samples showed good concordance in fluorescence intensity with standard RT-PCR (Pearson coefficients > 0.9). Клинические тесты с 36 образцами мазков из носа показали хорошее соответствие интенсивности флуоресценции стандартной ОТ-ПЦР (коэффициенты Пирсона > 0,9). Clinical tests with 36 samples of nasal swabs showed good agreement with the fluorescence intensity of standard RT-PCR (Pearson’s coefficients > 0.9).RT-PCR Клинические испытания 36 образцов мазков из носа показали хорошее совпадение интенсивности флуоресценции со стандартной ОТ-ПЦР (коэффициент Пирсона > 0,9). Clinical testing of 36 nasal swab specimens showed good agreement of fluorescence intensity with standard RT-PCR (Pearson’s coefficient > 0.9). In parallel with this work, various emerging biochemical methods (eg, plasma thermal cycling, amplification-free immunoassays, and nanobody functionalization assays) have shown their potential in POCT. However, due to the lack of a fully integrated and robust POCT platform, these methods inevitably require separate pre-processing procedures (e.g., RNA isolation44, incubation45 and washing46), which further complements the current work with these methods to implement advanced POCT functions with the required parameters. fetch-in-response-output performance. In this work, although the air pump used to activate the FAST valve is small enough to be integrated into a benchtop instrument (Fig. S9, S10), it still consumes significant power and generates noise. In principle, smaller form factor pneumatic pumps can be replaced by other means, such as the use of electromagnetic force or finger actuation. Further improvements may include, for example, adapting kits for different and specific biochemical assays, using new detection methods that do not require heating/cooling systems, thus providing a tool-free POCT platform for PCR applications. We believe that given that the FAST platform provides a way to manipulate liquids, we believe that the proposed FAST technology presents the potential to create a common platform not only for biomedical testing, but also for environmental monitoring, food quality testing, material and drug synthesis . .
The collection and use of human nasal swab specimens has been approved by the Ethics Committee of Zhejiang University First Affiliated Hospital (IIT20220330B). 36 nasal swab samples were collected, involving 16 adults < 30 years old, 7 adults > 40 years old, and 19 males, 17 females. 36 nasal swab samples were collected, involving 16 adults < 30 years old, 7 adults > 40 years old, and 19 males, 17 females. Было собрано 36 образцов мазков из носа, в которых приняли участие 16 взрослых < 30 лет, 7 взрослых старше 40 лет, 19 мужчин и 17 женщин. Thirty-six nasal swab specimens were collected from 16 adults < 30 years of age, 7 adults over 40 years of age, 19 men and 17 women. Demographic data are presented in Supplementary Table 3. Informed consent was obtained from all participants. All participants were suspected of having influenza and were tested voluntarily without compensation.
The FAST base and lid are made of polylactic acid (PLA) and printed by Ender 3 Pro 3D printer (Shenzhen Transcend 3D Technology Co., Ltd.). Double sided tape was purchased from Adhesives Research, Inc. Model 90880. PET film 100 µm thick was purchased from McMaster-Carr. Both the adhesive and the PET film were cut using the Silhouette Cameo 2 cutter from Silhouette America, Inc. The elastic film is made of PDMS material by injection molding. First, a 200 µm thick PET frame was cut using a laser system and glued to a 3 mm thick PMMA sheet using 100 µm double-sided adhesive tape. The PDMS precursor (Sylgard 184; Part A: Part B = 10:1, Dow Corning) was then poured into the mold and a glass rod was used to remove excess PDMS. After curing at 70° C. for 3 hours, the 300 μm thick PDMS film could be peeled off the mould.
Photos for versatile distribution, on-demand publishing and reliable performance are taken with a high-speed camera (Sony AX700 1000 fps). The orbital shaker used in the reliability test was purchased from SCILOGEX (SCI-O180). The air pressure is generated by an air compressor, and several digital precision pressure regulators are used to adjust the pressure value. The flow behavior testing process is as follows. A predetermined amount of fluid was injected into the test device and a high speed camera was used to record the flow behavior. Still images were then taken from videos of the flow behavior at fixed times, and the remaining area was calculated using Image-Pro Plus software, which was then multiplied by the camera depth to calculate the volume. Details of the flow behavior testing system can be found in Supplementary Figure S4.
Inject 50 µl of microbeads and 100 µl of deionized water into the vial mixing device. Mixed performance photographs were taken with a high speed camera every 0.1 seconds at pressures of 0.1 bar, 0.15 bar and 0.2 bar. Pixel information during the blending process can be obtained from these images using photo processing software (Photoshop CS6). And mixing efficiency can be achieved with the following Equation 53.
where M is the mixing efficiency, N is the total number of sample pixels, and ci and \(\bar{c}\) are the normalized and expected normalized concentrations. Mixing efficiency ranges from 0 (0%, unmixed) to 1 (100%, fully mixed). The results are shown in Supplementary Figure S6.
Real-time RT-PCR kit for IAV and IBV, including IAV and IBV RNA samples (cat. no. RR-0051-02/RR-0052-02, Liferiver, China), Tris-EDTA buffer (TE buffer no. B541019, Sangon Biotech, China), Positive Control RNA Purification Kit (Part No. Z-ME-0010, Liferiver, China) and GAPDH Solution (Part No. M591101, Sangon Biotech, China) are commercially available. The RNA purification kit includes a binding buffer, wash A, wash W, eluent, magnetic microbeads, and an acrylic carrier. IAV and IBV real-time RT-PCR kits include IFVA nucleic acid PCR detection mix and RT-PCR enzyme. Add 6 µl of AcrylCarrier and 20 µl of magnetic beads to 500 µl of binding buffer solution, shake well and then prepare the bead solution. Add 21 ml of ethanol to washes A and W, shake well to obtain solutions of washes A and W, respectively. Then, 18 µl of fluorescent PCR mixture with IFVA nucleic acid and 1 µl of RT-PCR enzyme were added to 1 µl of TE solution, shaken and centrifuged for several seconds, obtaining 20 µl of IAV and IBV primers.
Follow the following RNA purification procedure: (1) RNA adsorption. Pipette 526 µl of the pellet solution into a 1.5 ml centrifuge tube and add 150 µl of sample, then manually shake the tube up and down 10 times. Transfer 676 µl of the mixture to the affinity column and centrifuge at 1.88 x 104 g for 60 seconds. Subsequent drains are then discarded. (2) The first stage of washing. Add 500 µl of wash solution A to the affinity column, centrifuge at 1.88 x 104 g for 40 s, and discard the spent solution. This washing process was repeated twice. (3) the second stage of washing. Add 500 µl of wash solution W to the affinity column, centrifuge at 1.88×104 g for 15 s and discard the spent solution. This washing process was repeated twice. (4) Elution. Add 200 µl of eluate to the affinity column and centrifuge at 1.88 x 104 g for 2 min. (5) RT-PCR: The eluate was injected into 20 μl of the primer solution in a PCR tube, then the tube was placed in a real-time PCR test apparatus (SLAN-96P) to carry out the RT-PCR process. The entire detection process takes approximately 140 minutes (20 minutes for RNA purification and 120 minutes for PCR detection).
526 µl of bead solution, 1000 µl of wash solution A, 1000 µl of wash solution W, 200 µl of eluate and 20 µl of primer solution were preliminarily added and stored in chambers M, W1, W2, E and PCR detection chambers. Platform assembly. Then, 150 µl of the sample was pipetted into chamber M and the FAST-POCT platform was inserted into the test instrument shown in Supplementary Figure S9. After about 82 minutes, the test results were available.
Unless otherwise noted, all test results are presented as mean ± SD after a minimum of six replicates using only the FAST-POCT platform and biologically independent samples. No data was excluded from the analysis. The experiments are not random. The researchers were not blind to group tasks during the experiment.
For more information on study design, see the Nature Research Report abstract linked to this article.
Data supporting the results of this study are available in Supplementary Information. This article provides the original data.
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