Recent advances in centrifugal microfluidics for point-of-care testing

Abstract

Point-of-care testing (POCT) holds significant importance in the field of infectious disease prevention and control, as well as personalized precision medicine. The emerging microfluidics, capable of minimal reagent consumption, integration, and a high degree of automation, play a pivotal role in POCT. Centrifugal microfluidics, also termed lab-on-a-disc (LOAD), is a significant subfield of microfluidics that integrates crucial analytical steps onto a single chip, thereby optimizing the process and enabling high-throughput, automated analysis. By utilizing rotational mechanics to precisely control fluid dynamics without external pressure sources, centrifugal microfluidics facilitates swift operations ideal for urgent medical and field settings. This review provides a comprehensive overview of the latest advancements in centrifugal microfluidics for POCT, covering both theoretical principles and practical applications. We begin by introducing the fundamental operational principles, fluidic control mechanisms, and signal output detection methods. Subsequently, we delve into the typical applications of centrifugal microfluidic platforms in immunoassays, nucleic acid testing, antimicrobial susceptibility testing, and other tests. We also discuss the strengths and potential limitations of centrifugal microfluidic platforms, underscoring their transformative impact on traditional conventional procedures and their significant role in diagnostic practices.

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1 Introduction

Point-of-care testing (POCT) has become an invaluable asset in medical diagnostics, enabling rapid and convenient testing directly at the site.^1,2 By allowing tests to be performed bedside or in remote settings, POCT eliminates the delays associated with centralized laboratories and professional operation.^3,4 Its ability to provide immediate results significantly enhances diagnostic speed and accuracy, which is crucial for effective clinical decision-making and timely initiation of treatment. Moreover, the emergence of recent global pandemics, most notably COVID-19, has accelerated the demand for efficient diagnostic methods like POCT.^5,6 These widespread health crises have not only posed severe threats to public health but have also disrupted economies and altered social structures worldwide.^7,8 Therefore, there is an urgent need for advanced POCT platforms to address these challenges effectively. However, despite the vital role of POCT, existing platforms often face limitations in speed, accuracy, and accessibility, which can hinder timely detection and response.

To further enhance the efficiency and effectiveness of POCT, microfluidic technology is increasingly being recognized as a game-changer.^9,10 It integrates essential operations, such as sample preparation, reaction, separation, and detection, onto a single microscale chip, automating the entire process of biological, chemical, and medical analysis.^11–13 Microfluidic systems can be classified into two primary categories based on their fluid control mechanisms: active and passive microfluidic chips.^14,15 Passive microfluidics typically utilizes natural forces, such as surface tension, wettability, capillary action, and gravity, to drive fluid flow.¹⁶ Although this approach lowers the need for equipment, it demands high design and processing precision and offers limited flexibility in flow rate adjustment, making complex fluid control challenging. Active microfluidics utilizes external driving forces for precise fluid control, which can be categorized into pressure-driven, digital, magnetism-based, and centrifugal-driven types.¹⁷ Pressure-driven microfluidics employs external pressure sources to regulate fluid motion with adjustable flow rates.¹⁸ However, this approach often restricts the flexibility of flow paths and requires multiple pressure sources for multi-directional fluid control. Digital microfluidics exploits the electrowetting effect through electrodes to manipulate droplets, offering high integration and programmability but necessitating complex electrode arrays.¹⁹ This complexity may limit the size and precision of droplet manipulation, particularly when dealing with large sample volumes. Magnetic microfluidics relies on magnetic fields for non-contact fluid control; however, its reliance on magnetic materials limits the range of applicable samples. In comparison to the aforementioned technologies, centrifugal microfluidics or lab-on-a-disc (LOAD) enables intricate fluid control using a simple rotational device while eliminating the need for external pressure sources and thereby simplifying system design and reducing costs.²⁰ With its fast response time, it allows for quick fluid operations, making it particularly well-suited for applications requiring quick analysis. Given these advantages, centrifugal microfluidics is rapidly becoming a cornerstone technology in fields that demand both speed and scalability, revolutionizing traditional laboratory processes and significantly propelling the frontiers of scientific and analytical research. By offering rapid, on-site diagnostic functions, centrifugal microfluidic platforms significantly streamline POCT procedures, ensuring reliable and efficient testing available across diverse medical and field-based settings.^21,22

Previously, several reviews have introduced various applications of centrifugal microfluidic platforms,²³ including biomedical applications,²⁴ particle/cell separation,²⁵ immunoassays and nucleic acid testing.^21,26 However, there remains a dearth of comprehensive and systematic introductions to centrifugal microfluidic platforms, especially in POCT. Although Ducrée et al. (2007)²⁷ and Strohmeier et al. (2015)²⁰ have compiled relevant work, the rapid development of centrifugal microfluidic platforms necessitates an updated review of recent research. On account of this, this review systematically presents the latest advancements in centrifugal microfluidics for point-of-care diagnostics from principle, signal output to applications (Fig. 1). We first present the fundamental principles of centrifugal microfluidics and how structural design enables precise fluid control on centrifugal microfluidic platforms. Then we introduce different types of signal outputs, encompassing colorimetric, fluorescent, electrochemical, and distance measurements. Following this, we delve into centrifugal microfluidic platforms for immunoassays, nucleic acid testing, antimicrobial susceptibility testing, and other tests. Finally, this review concludes with a summary and outlook on the strengths and limitations of centrifugal microfluidic platforms.

Fig. 1 Schematic illustration of centrifugal microfluidics for point-of-care testing.

2 Centrifugal microfluidic analysis techniques

2.1 Fundamental principles of centrifugal microfluidics

Centrifugal microfluidics is a significant branch of microfluidic technology, specifically referring to the use of centrifugal force to drive the flow of small volumes of liquid.²⁰ This technology integrates basic operations found in biological and chemical fields onto a small, disc-shaped chip, often referred to as a lab-on-a-disc (LOAD). Typically, a centrifugal microfluidic chip resembles a small CD and comprises multiple microchannels, storage chambers, reaction chambers, and detection chambers.^28–31 By rotating the chip, the liquid within it is primarily driven by three types of forces: centrifugal force, Coriolis force, and Euler force, enabling various precise fluidic operations such as mixing, separation, filtration, and reaction.³² As shown in Fig. 2, these processes can be represented by the following formulas:

F_c = −mω²R

F_co = −2m[small omega, Greek, vector] × [small nu, Greek, vector]

The symbol F_c represents the centrifugal force, F_co denotes the Coriolis force, and F_e stands for the Euler force. The variable m represents the mass of the fluid, ω is the angular velocity of rotation, and r is the vector from the particle to the axis of rotation. The symbol R indicates the distance from the fluid particle to the center of rotation, while [small nu, Greek, vector] represents the rotational velocity of the object. Centrifugal force is generated by a rotational centrifugal field, and it acts in the direction away from the center of rotation. Specifically, in centrifugal microfluidic chips, the greater the rotational velocity, the stronger the centrifugal force, which in turn increases the force exerted on the fluid. This facilitates faster fluid flow and enhanced mixing efficiency. The Coriolis force is induced by the free movement of fluid within a centrifugal field, acting perpendicular to the fluid's velocity. This force can generate shear motion and vortices within the fluid. In centrifugal microfluidic chips, the Coriolis force aids in achieving more thorough mixing, dispersion, and flow path gating. The Euler force arises from the rotation of a rotating reference frame relative to a fixed reference frame. It represents the combined effect of centrifugal and Coriolis forces, influencing the fluid's velocity and direction. This force can be utilized to control the flow direction and distribution of the fluid, enabling more precise fluid manipulation. The Euler force is perpendicular to the centrifugal force, and its direction depends on the acceleration. This force only arises when the disk undergoes non-uniform circular motion. Typically, the interactions among these forces result in controllable fluid flow and mixing within centrifugal microfluidic chips, enabling precise control and manipulation of small fluid volumes. Designing and optimizing centrifugal microfluidic chips requires accounting for the interactions between centrifugal, Coriolis, and Euler forces, as well as the microchannel geometry and layout, to ensure efficient, precise, and reliable fluid control. Additionally, factors such as fluid properties (e.g., viscosity, density, surface tension) and the rotational speed and direction of the centrifugal field must also be taken into account.

Fig. 2 Schematic diagram of a centrifugal microfluidic chip and the three main forces generated by rotation. The centrifugal force is directed away from the center of rotation, while the Coriolis force acts perpendicular to the direction of velocity. Therefore, in centrifugal microfluidic chips, the rotation direction can be used to control liquid separation. The Euler force, which only exists in a non-uniform centrifugal field, is the combined effect of centrifugal and Coriolis forces. By controlling the centrifugal acceleration, the direction of fluid flow can be precisely controlled.

Madou and Duffy, among other researchers, characterized the flow rates of fluids in centrifugal microchannel structures and compared the results with centrifugal theory.^32,33 According to the continuity equation for incompressible fluid,^31,32 the average fluid velocity U can be expressed as:

S is the cross-sectional area through which the liquid flows, and Q is the volumetric flow rate. In microchannels, particularly in centrifugal microchannels, the calculation of flow velocity involves various factors, including the pressure gradient, channel geometry, and hydrodynamic resistance. For laminar, viscous, and incompressible flow, the formula for calculating the flow rate Q is:
where ΔP_c is the pressure gradient and R is the hydrodynamic resistance of the channel. The hydrodynamic resistance of the channel is determined by the cross-sectional geometry of the channel and can be calculated using the following formula:
where L, w, and h represent the length, width, and height of the channel.

In centrifugal microchannels, since the centrifugal force is the primary driving force, the pressure difference across the radial ends of the liquid-filled channel can be simplified to:

where r₁ and r₂ represent the distances from the fluid to the center of rotation at the closest and farthest points, respectively.

Thus, the average velocity of the fluid can be derived as follows:³³

2.2 Valve control on centrifugal microfluidic platforms

Microvalves are a crucial component in centrifugal microfluidic systems, which are essential for integrating multiple unit operations such as liquid transport, mixing, dispensing, metering, and separation.³⁴ The physicochemical properties of reagents involved need to be carefully considered in the design and selection of valves. Inappropriate valve selection can lead to evaporation or leakage of samples or reagents, potentially affecting downstream reactions. According to the principle of control, we divide centrifugal microfluidic valves into two categories: passive valves and active valves.³⁵ Passive valves typically rely on the inherent properties of the fluid for control, while active valves often incorporate external components.

2.2.1 Passive valves. Passive valves operate based on intrinsic parameters of the centrifugal system, such as centrifugal speed, acceleration, and rotational direction. In the default state, these valves are typically closed, and subsequently triggered by capillary, surface tension, or fluid pressure differences.³⁶ These valves control fluid flow through precise chip structure designs and generally rely on surface modifications of the chip channels, thus reducing the necessity for complex external instrumentation.³⁷
2.2.1.1 Capillary valves. The capillary valves are based on the competition between centrifugal force and capillary force: when the centrifugal pumping pressure is less than the capillary barrier, the fluid will not pass through the capillary valve.^38,39 Therefore, in practical applications, the capillary valve is mainly controlled by the increase or decrease of the speed (Fig. 3A-i). When the centrifugal force fails to overcome the capillary resistance, the capillary valve remains closed; conversely, when the capillary resistance is exceeded, the capillary valve opens. Thus, the capillary valve belongs to a high-pass valve. The value of capillary force can be calculated using the surface free energy in thermodynamics,⁴⁰ as follows:

ΔP_s = Cγ sin θ/A

where C is the circumference of the liquid in contact with the capillary, γ is the surface tension of the liquid, θ is the contact angle, and A is the cross-sectional area of the capillary. When the pressure ΔP_c at the liquid meniscus is less than the capillary force, the liquid halts at the expansion of this channel; however, if the pressure surpasses capillary resistance, the liquid can enter the expansion zone and continue to flow.⁴¹ It can be calculated that the burst frequency is:
where d_H = 4A/C is expressed as the hydraulic diameter or hydrodynamic diameter of the microchannel connecting the expansion zone, and ΔR represents the radial length of the liquid in the channel, while R̄ is the average value of R₁ and R₂.

Fig. 3 Principles of passive and active valves. (A) Capillary valve: (i) structural principle of the capillary valve structure; (ii) schematic diagram of the capillary stop valve (reprinted with permission from ref. 42). (B) Siphon valve: (i) principle of the structure of the pneumatic siphon valve; (ii) schematic diagram of the reciprocating flow pneumatic siphon valve (reprinted with permission from ref. 43). (C) Soluble membrane valve: (i) schematic diagram of the structure of the soluble membrane valve; (ii) operation of the digital pulse actuated dissolvable film valve (reprinted with permission from ref. 44). (D) Paraffin valve: (i) structure of the paraffin valve structure; (ii) schematic of the thermal drive oil valve (reprinted with permission from ref. 45). (E) Laser valve: (i) schematic diagram of the structure of the laser valve; (ii) laser-actuated valve opening (reprinted with permission from ref. 46). (F) Diaphragm valve: (i) schematic diagram of the diaphragm valve structure; (ii) concept illustration of the electric drive diaphragm valve (reprinted with permission from ref. 47).

From the discussion above, it is evident that the dimensions of the microchannel in the capillary valve, its radial position, the contact angle of liquid, and surface tension all play a decisive role in determining the breakthrough rotational speed.^10,48 By controlling these key parameters, it is possible to achieve either serial or parallel configurations of multiple capillary valves.⁴⁹ However, when the liquid contact angle decreases below 45°, the stability of capillary valves with respect to wetting liquids diminishes, leading to a reduced breakthrough rotational speed. In response, researchers have proposed the design of hydrophobic valves by applying a hydrophobic layer to the channels to increase the liquid contact angle and enhance the stability of valve control on the chip.^50,51 The valve control can also be stabilized by sudden narrowing or sudden widening of the channel design, which increases the breakthrough pressure by reducing the cross-sectional area of the fluid operation and reduces the fluid operation speed, respectively, to achieve the valve function of resisting low speed resistance. Based on this principle, our group has utilized T-shaped capillary shut-off valves in the measurement channels for rapid determination of phase diagrams (Fig. 3A-ii). Sample autonomously flowed into the metering chamber under capillary action, where the capillary stop valve in the metering channel effectively prevents further diffusion, thereby facilitating precise and controlled quantification.⁴² On the basis of the original T-shaped capillary stop valve, Chang's group has developed a beveled capillary stop valve that reduces the backflow commonly observed in traditional designs, ensuring unidirectional and complete flow of samples.⁵²

2.2.1.2 Siphon valves. Siphon valves feature a U-shaped channel divided into rising and falling sections, typically rendered hydrophilic to allow capillary forces to activate the valve effectively at reduced rotational speeds (Fig. 3B-i).¹⁵ At sufficiently high rotational speeds, the centrifugal force exceeds the capillary force, preventing the liquid in the upstream chamber from surpassing the siphon high point, thus keeping the valve closed. However, when the rotational speed drops below a critical threshold, the capillary force begins to dominate, causing the liquid in the upstream chamber to fill the siphon channel and surpass the siphon high point.⁵³ Upon subsequent activation of the centrifuge, the siphon valve opens, allowing the liquid from the upstream chamber to flow into the downstream chamber via the siphon channel. Therefore, siphon valves are classified as low-pass valves, triggered by a reduction in flow rate. On this basis Xu and colleagues proposed a Euler force-assisted siphon valve, where the ascending and descending sections are activated by Euler and capillary forces, respectively. Under fixed channel dimensions and angles, siphon channels of varying lengths can be triggered by different Euler forces, making the sequential release of liquids more convenient and flexible.⁵⁴ Nevertheless, conventional siphon valves require hydrophilic modification of the siphon channel, addressing issues related to the selection of hydrophilic reagents, hydrophilic retention time, and reproducibility. To mitigate the impact of hydrophilic reagents, researchers have introduced a series of upgraded siphon valves, including overflow siphon valves, pneumatic siphon valves, and thermal pneumatic siphon valves, thereby enhancing the versatility of siphon valves in centrifugal microfluidics.^55,56 For instance, Godino et al. designed a pneumatic-triggered siphon valve consisting of an inlet channel, an outlet siphon channel, and a compression chamber, all interconnected.⁵⁷ At a given rotational frequency, the liquid levels in the inlet and siphon channels are positioned below the peak of the siphon tube due to fluid statics, while the liquid level in the compression chamber is lower due to centrifugal forces. Upon reducing the rotational speed, the trapped gas expands, acting as a variable-frequency pump, thereby pushing the liquid from the ballast chamber into the inlet and siphon channels. Furthermore, our group propose a centrifugo-pneumatic reciprocating flow coupled with spatial confinement strategy (Fig. 3B-ii), allowing for bidirectional flow of reaction liquids.⁴³ Additionally, to enhance the flexibility of flow control, allowing for control of the flow even after the siphon valve filled with liquid, Xu et al. proposed an interruptible siphon valve.⁵⁸ This technology facilitated on-demand activation of the siphon valve by controlling the centrifugal speed. Next, by increasing the rotational speed, the centrifugal force acting on the fluid in the siphon channel was enhanced. This allowed air to enter through an aperture in the interruptible siphon valve, cutting off the fluid flow and effectively shutting down the siphon effect.
2.2.1.3 Dissolvable film valves. Dissolvable films (DFs) are thin films made from specialized materials that gradually disintegrate upon exposure to specific liquids (Fig. 3C-i). Generally, the opening and closing of DF valves are independent of manufacturing errors, with relatively simple chip structures and lower fabrication complexity.⁵⁹ For instance, Gorkin III and colleagues achieved breakthrough frequencies nearly ten times higher than conventional capillary valves by combining rotationally driven gas barriers and flow control with DF valves.⁶⁰ At low rotational speeds, the gas–liquid interface formed by trapped air prevents the liquid column stabilized by surface tension from wetting the DFs. Conversely, high rotational speeds disrupt the metastable gas–liquid interface equilibrium, allowing the liquid to wet the DF and open the valve. Single DF valves are often insufficient for multiple fluid control, leading to the development of an event-triggered system with multiple DFs.⁶¹ Typically, such event-triggered systems employ a control film (CF) to induce the release of liquid through a load film (LF). To be more specific, Mishra and colleagues proposed a pulse-driven valve that employed the CF and LF to establish pneumatic chambers sealed by DFs (Fig. 3C-ii). The system remains closed at high rotational speeds. Upon reducing the speed, the assisting liquid dissolves the CF, allowing the sample to enter the valve. Upon re-accelerating, the sample moves to the end chamber, and once the LF is wetted, the valve opens.⁴⁴

2.2.2 Active valves. Active valves rely on external excitation or energy to operate, and usually require external equipment to initiate, halt, or regulate fluid flow within the micro-scale flow channel.⁶² The design and implementation of active valves usually involves microelectromechanical system technology or other micro-drive technologies to achieve high precision and response speed, including paraffin valves, laser valves and diaphragm valves.⁶³
2.2.2.1 Paraffin valves. Paraffin valves operate by leveraging the phase change characteristics of paraffin, which transitions from solid to liquid across different temperatures to regulate fluid flow.⁶⁴ At room temperature, paraffin remains solid, maintaining the valve in a closed position. By applying localized heat with lasers or hot air guns, paraffin quickly melts, thereby opening the valve structure during centrifugation (Fig. 3D-i). It was reported that paraffin valves maintain stability without leakage under centrifugal forces up to 403.0 ± 7.6 kPa, with valve operation unaffected by rotational speed, sequence, sample type, or substrate material characteristics.⁶⁵

In addition to laser heating, some groups have employed handheld or wirelessly controlled heat sources as alternatives to infrared sources for melting paraffin, aiming to reduce reaction costs and improve convenience.⁶⁵ Besides, Wang's group used heating resistance and wireless transmission technology to develop the liquid sequential loading structure and liquid flow logic control structure.⁶⁶ This setup benefits from the co-localization of paraffin valves and heating resistors, allowing for valve control during chip rotation without the need for precise positioning. Moreover, the melting process of paraffin to open fluid pathways can be reversed, and the valve can close again if paraffin melts to block the channel and then re-solidifies.⁶⁷ In addition to traditional solid paraffin, researchers are also exploring similar lipophilic and hydrophobic materials for valve control.⁶⁸ For instance, Rowlands and colleagues designed a thermally driven valve (Fig. 3D-ii) with an integrated heat source within the chip, using olive oil as an expansion medium and heating wire as the heat source.⁴⁵ The expansion of the oil upon heating can completely block the channel, halting liquid flow. Compared to traditional paraffin and thermal-driven valves, it features simpler components, requires no additional cleanroom, and is more cost-effective. While thermal-driven valves, such as those utilizing paraffin or olive oil, are widely used for fluid control in microfluidic systems, other temperature-sensitive materials, such as ice, also show great promise in valve control. One such approach involves the use of ice-valves, which utilize the freezing and melting of low-temperature liquids to regulate fluid flow. For example, Amasia et al. demonstrated the first utilization of ice-valving in an integrated centrifugal microfluidic system, employing it to seal the thermocycling chamber and minimize fluid loss from evaporation.⁶⁹ This method, while distinct from traditional thermal-driven systems, opens new possibilities for precise control, especially in applications where reversible changes in valve state or temperature-driven switching are required.

2.2.2.2 Laser valves. Laser valves achieve precise fluid control through laser-triggered activation, characterized by non-contact operation.⁷⁰ Compared to paraffin valves, laser valves offer faster response times and effectively reduce the risks of cross-contamination and leakage (Fig. 3E-i). Woolf et al. applied carbon powder within microchannels, which utilized the differences in thermal absorption properties between the focused laser spot and the material to achieve simple and efficient valve control (Fig. 3E-ii).⁴⁶ The transparent surface layer exhibited high transparency and low thermal absorption efficiency under wavelengths above 400 nm, while the black coating enhanced the conversion of light energy into thermal energy. This setup configuration generated quick localized heating at the illuminated area, causing the valve to open by melting, and then closed when the heating source is withdrawn. This novel approach simplified the design of centrifugal active valves, increased the number of integrated unit operations, and automated complex fluid control. Park et al. used laser-irradiated ferrowax microvalves to automatically control fluid transfer on the disc.⁷¹ It enabled the automated multiple standard addition process without the need for any manual interventions.
2.2.2.3 Diaphragm valves. Diaphragm valves depend on the deformation of the elastic membrane (Fig. 3F-i). They utilize external force to induce membrane deformation, effectively sealing the channel and closing the valve.⁷² Upon withdrawal of the force, the membrane resumes the original configuration, thereby reopening the valve. Diaphragm valves can be categorized into two types based on the nature of the external force applied: manual and mechanical. Manual diaphragm valves necessitate manual adjustment to regulate fluid flow. For example, Hoang et al. designed a screw valve for channel isolation by rotating the valve with a screwdriver to open or close the flow path.⁷³ Mechanical diaphragm valves operate via electric, pneumatic or hydraulic drive systems without the need for external intervention. For instance, Cho's group demonstrated a centrifugal microfluidic platform integrating individually addressable diaphragm valves that enable the reversible and thermally stable actuation of multiple valves with unprecedented ease and robustness.⁷⁴ These valves could be controlled via computer to address and press the valve while rotating it by a specific angle, applying pressure to the diaphragm and sealing the microchannel. Programmable electromagnetic actuation can further simplify chip operations while streamlining the control processes. For example, Xu and colleagues designed an electromagnetic-triggered column valve composed of a metal pin and pressure-sensitive adhesive tape.⁷⁵ To reduce the number of localized valve components and simplify chip fabrication, our group proposed a centrifugal microfluidic chip based on an online dual-valve system, integrating puncture valves and reversible active valves (Fig. 3F-ii).⁴⁷ This system, supported by electrical drive, effectively and reliably controls fluid introduction, retention, and flow.

2.2.3 Pros and cons of valves. In summary, passive valves control fluid flow based on surface tension or physical barriers and can be driven by variations in rotational speed. In contrast, active valves typically involve external devices to control valve operation, with higher adjustability. Despite their widespread application and effective fluid control, these valves are still limited in practical applications (Table 1).³⁵ Passive valves rely primarily on inherent forces within the chip to manage fluid flow, which limits control precision and results in slower response times. Active valves, on the other hand, often require higher equipment costs and increased system energy consumption. Specifically, passive valves like capillary^42,76 and siphon valves are influenced by physical properties of the fluid, such as viscosity and surface tension, which can limit control and result in longer response times. Moreover, some passive valves, like dissolvable film valves,⁷⁷ are sacrificial and cannot be reopened once used, further limiting their applicability. Siphon valves additionally perform poorly with high-viscosity liquids due to their dependence on siphon force. Active valves, such as paraffin^78,79 and diaphragm valves,^75,80,81 also face challenges. They require specific materials that can significantly affect valve response times and potentially interact with reagents or processes negatively, for instance, leaving residues from external heat sources. Laser valves offer precision but come with high equipment costs and stringent requirements for system design and maintenance. In addition, diaphragm valves face limitations such as increased system complexity from the need for precise external actuation mechanisms and potential performance degradation over time due to membrane fatigue. Optionally, elastic membrane valves operate without the need for external controls, offering a simpler, more integrated approach. For example, Hwang et al. presented elastomeric membrane valves integrated into a centrifugal microfluidic platform, where fluid flow, dependent on disc rotation speed and membrane thickness, can be precisely controlled by adjusting the disc motion.⁸² Ultimately, while each valve design offers distinct advantages, challenges such as system complexity, stability, and flow control limitations remain, indicating that no single valve type can comprehensively meet all the demands of centrifugal microfluidic systems.

Table 1 Performance comparison of different valves

Valve	Advantage	Disadvantage
Capillary valve	Simple design, automatic fluid control	Susceptibility to clogging, dependence on surface modification, fluid properties and channel sizes, lack of stability and control accuracy
Siphon valve	Suitability for continuous or timed fluid flow	Larger physical space, difficulty in transporting high-viscosity fluids
Dissolvable film valve	Simple and flexible, easy to control	Difficulty in reuse, high breakthrough speed
Laser valve	Fast response speed, high control accuracy, contactless control	High costs, equipment specificity, potential reagent contamination
Paraffin wax valve	Reusability and independence of speed and sample type	Limited response speed, equipment specificity, potential reagent contamination
Diaphragm valve	Reversible, high control accuracy	Difficulty of heterogeneous manufacturing, increased costs

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Exploration of new materials and valve designs continues to advance. Aeinehvand et al. proposed the reversible thermo-pneumatic valve combined pneumatic valves with active control, which operate on the thermal expansion or contraction of air and the deflection of a latex elastic membrane (Fig. 4A).⁸³ It featured a gas-impermeable structure with a latex-sealed air chamber and a liquid transition chamber that optimizes thermal energy use, and it either sealed or reopened an inlet by heating or cooling a trapped air volume. In addition, as an alternative to hydrophilic modification, Suzuki et al. used the wettability changes in conductive polymers, like polypyrrole (PPy), to modify the electrode potential of the valve, enabling the PPy membrane to exhibit varying polarities (Fig. 4B).⁸⁴ This makes the valve an active valve, as its operation is driven by an external electric potential. Specifically, when a reducing potential is applied to the valve electrode, the PPy membrane on the electrode surface exhibits hydrophilicity due to the reorientation of dopant molecules, allowing the solution to flow smoothly.

Fig. 4 Novel valves and mixing valves. (A) Reversible hot air valve (reprinted with permission from ref. 83). (i) Principle of the structure of the reversible hot air valve. (ii) The state of the reversible hot air valve controlled by temperature. (B) Switchable microvalves employing a conducting polymer (reprinted with permission from ref. 84). (i) Schematic diagram of the switchable micro-valve composed of the PPy membrane. (ii) Switchable micro-valve controlled by the hydrophilicity of the channel through electronic difference. (iii) Working principle of the suction pump and the injection pump. (C) Mixing valve composed of the siphon valve and laser valve (reprinted with permission from ref. 85). (i) A three-dimensional and two-dimensional illustration of the unit valve control structure. (ii) The actual image of the disc before and after the crystal purple solution is twice diluted six times. (D) Working principle of the mixing valve with the capillary valve, gravity valve and Coriolis force (reprinted with permission from ref. 86).

A single valve often falls short of achieving precise control, especially in the case of multiple fluids. For comprehensive fluid control, researchers often employ a combination of multiple valves to ensure consistent and stable fluid management. Park et al. combined siphon valves and laser valves to achieve fully automated continuous dilution on a centrifugal microfluidic chip (Fig. 4C).⁸⁵ The accuracy of this dilution system was verified through continuous experiments involving six sequential two-fold and ten-fold dilutions, with the entire dilution process completed within 5 minutes. Besides, Wang et al. combined gravity valves, capillary valves, and Coriolis switching valves to design a centrifugal solid-phase extraction system (Fig. 4D).⁸⁶ In this application, the Coriolis force is primarily used for flow control within the centrifugal system. When the centrifugal platform rotates clockwise, the Coriolis force guides the liquid sample into the right chamber of the filter core; conversely, when rotating counterclockwise, the liquid sample enters the left chamber of the filter core.

As shown in Table 2, the typical examples of various valves are listed to provide a comparative discussion. It facilitates a deeper understanding of operational principles and application-specific advantages of each valve type, highlighting how different mechanisms cater to unique analytical requirements in microfluidic systems. This comparative analysis not only clarifies the functional distinctions between the valves but also aids in selecting the most appropriate valve type for specific applications, optimizing performance and efficiency in diagnostic and analytical procedures. As microfluidic technology continues to evolve, the design and functionality of valve systems are expected to be further refined, enhancing control accuracy and response speed for complex fluids. Additionally, the development of intelligent fluid control systems is anticipated. These systems will be capable of more flexibly adjusting fluid paths and flow rates to accommodate changing conditions.

Table 2 Classification of valves and their specifications on centrifugal microfluidics

Type	Valve	Maximum resistance (RPM)	Response time	Additional treatment	Extra equipment	Ref.
Passive	Trap/hydrophilic valve	818 ± 35/803 ± 39	—	Nanosecond and femtosecond lasers treatment	—	41
	Capillary valve	4000	5–10 s	Superhydrophobic modification	—	39
	Microchannels of capillary valve	1500	1 s	Hydrophobic modification	—	76
	Capillary stop valves	6000	—	—	—	42
	Hydrophilic and hydrophobic switchable valve	—	1–5 s	Polypyrrole modification	—	84
	Hybrid siphon valve	3000	—	Hydrophilic modification	—	55
	Interruptible siphon valve	3000	—	Hydrophilic modification	—	58
	Dissolvable films valve	3000	10 s	Water-dissolvable films integration	—	60
	Digital pulse-actuated dissolvable film valves	2400	30–40 s	Dissolvable films integration	—	44
	Selective membrane valve	4200	—	Solvent-selective lipophilic/hydrophobic membrane loading	—	68
	Event-triggered dissolvable film valve	2400	—	Dissolvable film integration	—	77
	Elastomeric membrane valve	600	—	Elastomeric membrane integration		82
Active	Wax valve	2000	—	Sealing material and wax loading	Hot-air gun	78
	Wax valve	1000	20 s	Paraffin wax microspheres loading	Heating resistors	66
	Multi-use wax valves	6500	6 s	Ferrowax integration	Magnet	79
	Thermally-actuated microfluidic membrane valve	6000	9 s	Olive oil loading	Resistance wire for heating	45
	Laser burst valve	10 000	—	—	Laser diode	70
	Ferrowax microvalve	—	—	Ferrowax integration	Laser module	85
	Optically-controlled closable microvalves	—	2.5 s	—	Laser diode	46
	Elastic reversible valves	7500	—	Elastic barrier integration	—	80
	Reversible and thermally stable diaphragm valve	6000	2 s	—	Computer-controlled valve actuation system	74
	Electromagnet-triggered pillar valve and capillary valve	3000	—	Metal pin and pressure sensitive adhesive tape integration	—	75
	Puncture valve (PV) and reversible active valve (RAV)	1000	—	—	Spindle drive motor and linear motor	47
	Reversible thermo-pneumatic valve	2500	10–23 s	—	Heat gun and infrared thermometer	83
	Addressable electrowetting valve	1200	1 s	Metal electrode integration	PCB board integrating WiFi module	81

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2.3 Fluid control on centrifugal microfluidic platforms

The rapid and accurate aliquot of samples and reagents is a key part of the analysis, and the quantity of reagents often has a significant impact on test results.^56,87 Accurate quantification not only ensures that chemical reactions occur under optimal conditions but also guarantees the reliability and reproducibility of results. This precision is particularly critical in fields like diagnostics, where the correct reagent concentration is directly linked to the interpretation of test results and subsequent medical decisions.

The quantity of reagents loaded onto the platform can be determined by the volume of the chambers or channels. By regulating the volume of chambers, which are completely filled by external forces such as centrifugation, precise quantification is achieved. Generally, a metering structure comprises an inlet connection channel, at least one metering chamber with a fixed volume, and an overflow that directs any excess volume to a waste chamber. For example, Steigert et al. presented a metering structure with two capillary burst valves, one at the outlet of the metering chamber and the other at the end of the overflow channel.⁸⁸ The capillary burst valve at the overflow outlet of the chamber is more readily activated, allowing excess liquid to be transferred to the waste chamber in advance. Meanwhile, the liquid in the metering chamber can be subsequently transferred to the detection chamber for quantitative analysis. Furthermore, they displayed siphon-based metering on centrifugal microfluidics for rapid colorimetric assays in human whole blood.⁸⁹ By minimizing manufacturing complexity, the structure could theoretically operate at high frequencies, enabling rapid sedimentation and meniscus flattening, significantly improving the metering precision. In these one-stage aliquoting platforms, the liquid volume is metered directly in the final reaction chambers. However, a potential issue is that pre-stored reagents may impact the metered volume, and dissolved reagents could transfer between chambers during metering, resulting in cross-contamination. Given this, Mark et al. proposed a two-stage aliquoting platform with a wide upstream channel, a narrow connecting channel and an unvented downstream receiving chamber.⁹⁰ This approach enabled precise measurement, independent of the volume of reagents pre-stored in the receiving chamber.⁹¹ For high throughput distribution, Schwemmer et al. introduced a centrifugo-pneumatic multi-liquid aliquoting designed for parallel aliquoting and pairwise combination of multiple liquids (Fig. 5A).⁹² At high rotational speeds, the air in the pneumatic chamber is compressed to achieve liquid metering within the metering chamber. Upon a rapid decrease in rotational speed, the compressed air pushes the metered liquid into the mixing chamber, thereby integrating liquid metering and the dispensing of multiple liquids. In addition to platforms designed for equal aliquoting of reagents, there are also systems with chambers of varying volumes that facilitate the quantification of different sample volumes, thereby generating concentration gradients. By precisely regulating the volumes of the chambers to create concentration gradients, it allows for continuous variation in concentration within a chip, thereby enhancing the accuracy and flexibility of the tests. For example, Li et al. designed a centrifugal microfluidic-based gradient generator for 25 reaction conditions with different concentration ratios within 10 min.⁴² In the system, the quantification channels are hydrophilic only at the bottom, with the other three sides being hydrophobic, allowing the solution to be spontaneously drawn in via capillary action. Upon entry, the channel abruptly widens at its end, forming a capillary stop valve that halts further movement of the solution, thereby enabling rapid quantification. Two different reagents are introduced into channels on the centrifugal microfluidic platform via centrifugal force and combine to form 25 different ratio solutions, quickly generating a concentration gradient. Further advancing this concept, our group proposed a novel multi-reagent synchronous dispensing technique on centrifugal microfluidics with a simple design (Fig. 5B).²² At low centrifugal speeds, following the addition of samples or reagents, multiple liquids are directed into several subdivision chambers. At this stage, the centrifugal force is less than the resistance of the downstream valve in the subdivided chamber, causing the valve to close. The excess liquid is directed to an overflow collection chamber or downstream waste chamber. Subsequently, through high-speed centrifugation, the liquid breaks through the valve, and the quantitative liquid is sequentially transferred to the downstream reaction chamber.

Fig. 5 Fluid quantification and flow path switching. (A) Multi-reagent synchronous quantification and aliquot platform based on trap (reprinted with permission from ref. 92). (B) Quantification of centrifugal difference and concentration gradient formation chip based on the capillary valve (reprinted with permission from ref. 22). (C) Flow path switched by adjusting the direction of rotation in the centrifugal microfluidic chip (reprinted with permission from ref. 56). (D) Centrifugal microfluidic platform with two rotating shafts for sequential releasing (reprinted with permission from ref. 93).

To minimize cross-contamination and enhance the adaptability of systems, two-stage aliquoting platforms are commonly employed in the initial stages. In this approach, the liquid is first aliquoted into an intermediate chamber, where it is measured and stabilized before being transferred to the final reaction chamber. This method helps to isolate the different stages of liquid handling, reducing the likelihood of unintended fluid transfer between chambers and ensuring more accurate quantification. However, as previously mentioned, current quantitative platforms largely depend on passive valves and chamber sizes, yet their stability and repeatability require further refinement. Precise design and manufacturing of chambers with varying volumes are essential to ensure accurate liquid metering. However, inconsistencies in chamber volumes can occur due to manufacturing tolerances and design complexities, which can undermine the precision of the quantification process. Therefore, improving both the design accuracy and operational stability of these systems is crucial for achieving reliable and consistent results.

Fluid manipulation on centrifugal microfluidic platforms greatly improves the accuracy and efficiency of testing, and realizes the automation and modularization of complex processes by precisely manipulating fluids on a microscale. In the process of fluid control, liquids can be selectively transferred between various chambers or channels through the physical valves mentioned earlier. Alternatively, it can also be achieved by leveraging techniques such as Coriolis forces, acceleration disparities, or meticulously designed structures. As shown in Fig. 5C, our group proposed a pneumatic-triggered siphon that controls fluid release.⁵⁶ During gradual centrifugation, the compressed pneumatic chamber pushes fluid back, preventing it from passing the siphon valve. In rapid deceleration, the chamber quickly releases air pressure, causing backflow that forces some fluid through the siphon valve. After re-centrifugation, the fluid flows downstream through the siphon tube for controlled release. A similar pneumatic siphon is used in the elution collection chamber to mix nucleic acid molecules uniformly before they enter the downstream distribution chamber. Chen et al. proposed a centrifugal microfluidic platform based on siphon (Fig. 5D).⁹³ By adjusting the angle of the microfluidic chip on the platform, they controlled the centrifugal force acting on the liquid, allowing for sequential sample loading. Additionally, varying the lengths of the siphon channels enhanced the stability of the process.

2.4 Signal detection on centrifugal microfluidic platforms

Signal detection on centrifugal microfluidic platforms encompasses a range of methods designed to accurately detect and analyze signals in various assays.³⁰ These platforms are uniquely designed to perform various analytical functions efficiently, utilizing signal detection methods, such as colorimetric, fluorescence, electrochemical, and distance-based techniques.²⁰ We delve into how each detection technique functions within centrifugal microfluidic systems, discussing their operational principles, typical applications, and integration strategies that enhance diagnostic accuracy and throughput.

2.4.1 Colorimetric detection. Colorimetric detection is a chemical analysis method used for qualitative or quantitative analysis based on color changes.^94–96 This method is simple, intuitive, and cost-effective, making it suitable for various in vitro diagnostic applications.^97–99 Colorimetric detection is based on Beer's Law, analyzing the concentration of chemical substances by measuring or observing the intensity of a specific color in the solution. Colorimetric detection relies on the characteristics of color changes to analyze target analytes. It can achieve qualitative or semi-quantitative results by observing the appearance or disappearance of color, or changes in color intensity. This method requires no additional signal triggers, relying directly on absorbance or visual observation of color differences to obtain results, thereby eliminating the need for complex signal processing equipment.^71,100,101 As a result, colorimetric detection-based POCT platforms have garnered increasing research interest.¹⁰²

Currently, commonly used colorimetric detection methods are based on the color change reaction of gold nanoparticles (AuNPs). By observing the color changes in the solution and analyzing the color differences, semi-quantitative detection of the analyte can be performed. For instance, Zhou et al. employed AuNPs for colorimetric detection to measure the concentration of heavy metal ions in samples.¹⁰³ The AuNPs under high NaCl conditions cause the solution to change from red to blue in the presence of heavy metals. In addition to performing colorimetric detection in solution, gold nanoparticle-based colorimetry can be significantly enhanced by combining it with lateral flow immunoassay (LFIA), which simplifies the experimental process, improves detection limits, and reduces material consumption. Bao et al. developed a method combining gold nanoparticle-based colorimetry with LFIA, using gold nanoparticles on test strips, which greatly reduced the limit of detection (LOD).¹⁰⁴ Besides, Zhang et al. developed bimetallic Ag-Au urchin-like hollow nanospheres to enhance the colorimetric brightness of AuNPs, thereby improving the signal strength.¹⁰⁵ However, these methods lack the capability to handle complex samples and multiple reagents, limiting their application to simple sample detection. Xu et al. developed a centrifugal microfluidic platform, integrating a compatible LFIA onto the platform.¹⁰⁶ Through the centrifugal system, multiple reagents are sequentially introduced, and the final result is displayed via color changes on the test strips. Both the detection limit and sensitivity were higher than traditional LFIA, achieving a complete “sample-to-result” workflow (Fig. 6A). Additionally, Paust et al. proposed a flow control method using centrifugal microfluidic technology for lateral flow strips.¹⁰⁷ By controlling the flow with centrifugal force, the reagents and the test strips interacted more thoroughly, making the color development on the LFIA more stable within the centrifugal system.

Fig. 6 Signal detection on centrifugal microfluidics. (A) Schematic of the ECLFIA system with colorimetric detection (reprinted with permission from ref. 108). (B) Principle of fluorescence immunoassays on centrifugal microfluidics (reprinted with permission from ref. 109). (C) Composition diagram of the centrifugal microfluidic chip and scheme of the sandwich sensing detection strategy with electrochemistry (reprinted with permission from ref. 110). (D) A distance-based bacterial proliferation sensor with naked-eye observation (reprinted with permission from ref. 111).

2.4.2 Fluorescence detection. Fluorescence detection is a technique that utilizes the fluorescence phenomenon for substance analysis, widely applied in fields such as biology, chemistry, medicine, and environmental science.¹¹² This method identifies and quantitatively analyzes specific molecules in a sample by measuring the emitted fluorescence. Depending on the type of fluorescent dye used, fluorescence detection can be categorized into organic fluorescent dyes, inorganic fluorescent substances, and biological fluorescent proteins.

Organic fluorescent dyes are the most commonly used fluorescent probes, including dyes such as the Cy series and rhodamine, which offer good photostability and chemical stability. Due to their high sensitivity and tunable emission wavelengths, they are widely used in biomedical and materials science research. For example, Phaneuf et al. utilized Cy5 dye-labeled biotinylated oligonucleotides and short complementary quencher strand chains to achieve a fluorescence-based nucleic acid enzyme assay.¹¹³ Similarly, He et al. employed fluorescence resonance energy transfer between rhodamine B and AuNPs.¹¹⁴ However, these methods currently face issues such as multiple cleaning steps, complex chemical modifications, and high costs. Kong et al. proposed a novel centrifugal microfluidic chip-based immunofluorescence analysis system to simplify the immunoassay process (Fig. 6B).¹⁰⁹ By using centrifugal force to ensure more thorough contact between fluorescence microsphere-labeled capture antibodies and biomarkers, this system enables rapid and cost-effective protein diagnostics.

Inorganic fluorescent substances are phosphors doped with rare earth metal ions in an inorganic matrix.¹¹⁵ Compared to organic fluorescent dyes, inorganic fluorescent substances can achieve broader excitation spectra by doping with different rare earth ions.¹¹⁶ Quantum dots, as a next-generation inorganic fluorescent material, not only offer broad excitation spectra like the previous generation of inorganic fluorescent substances but also feature high fluorescence intensity and a wide range of emission colors, making them suitable for complex multiplex assays.^117,118 Koh et al. achieved detection of botulinum toxin with fluorescent quantum dots, demonstrating advantages in automation, sensitivity, and detection time compared to the gold standard mouse bioassay.¹¹⁹ Riegger et al. utilized quantum dots loaded on a centrifugal microfluidic platform to enable fully automated detection of hepatitis A and tetanus.¹²⁰

Another type of fluorescent dye is biological fluorescent proteins, including green fluorescent protein (GFP) and red fluorescent protein (RFP).^121–123 Compared to the aforementioned fluorescent dyes, biological fluorescent proteins are naturally occurring and generally exhibit better biocompatibility with cells and tissues. By selecting different colors of fluorescent proteins, multiplex labeling can be achieved.^124,125 Additionally, fluorescent proteins can be directly tagged to target proteins using genetic engineering techniques. For example, Shin et al. used cell-free protein synthesis reagents and template DNA encoding green fluorescent protein to perform cell-free protein synthesis within droplets.¹²⁶ To further enhance detection comprehensiveness and specificity, multi-target detection was achieved by introducing quadruple fluorescence imaging. This approach used green and red fluorescent proteins along with AttoRho 101 and Cy5 fluorescent dyes to detect different targets and sequences, offering significant potential for advanced ddPCR applications.¹²⁷ Additionally, Salin et al. developed a fluorescence protein detection platform based on centrifugal microfluidics. This platform enables rapid on-site pre-concentration and screening of organic pollutants, while minimizing sample loss and contamination.¹²⁸

Centrifugal microfluidic platforms utilizing fluorescence detection are trending towards miniaturization and increased convenience to suit practical usage scenarios. For instance, Dong et al. utilized a light-emitting diode with a wavelength around 485 nm, coupled with optical filters and a dichroic beam splitter to illuminate reagents and collect fluorescence signals.¹¹⁴ Then they used a photodiode combined with filters for detection, designing a high-sensitivity, real-time centrifugal microfluidic chip for multiplex detection. To obtain real-time detection data while the chip is spinning, Ukita et al. applied stroboscopic technology to fluorescence signal detection, enabling imaging of fluorescent particles in the microfluidic disk at a rotational speed of 3000 rpm.¹²⁹

2.4.3 Electrochemical detection. Electrochemical detection is a method based on the electrochemical properties and their changes, involving the measurement of variations in electrical quantities such as potential, conductivity, current, and charge to qualitatively and quantitatively analyze components. Electrochemical sensing on centrifugal microfluidic platforms was first proposed in 1982,¹³⁰ and subsequently, electrical signal transmission gradually integrated into microfluidic platforms equipped with electrodes.¹³¹

Amperometry is the most commonly used electrochemical detection technique. In amperometry, a constant potential is applied to a working electrode, and the current–time trace resulting from the oxidation or reduction of electroactive substances is recorded to directly or indirectly detect the target analyte. When the target analyte is not electroactive, traditional amperometric methods cannot directly measure the signal from these biomarkers. Rattanarat et al. utilized screen-printed carbon paste electrodes modified with graphene–polyaniline nanocomposites to indirectly detect non-electroactive biological analytes.¹³² They integrated the electrodes into the centrifugal platform to enhance glucose detection. Glucose solution and enzyme solution were loaded into separate reservoirs, then mixed in a spiral channel. Within 7.5 minutes, an enzymatic reaction produced electrochemically active hydrogen peroxide, which was subsequently measured to determine the stoichiometric amount of glucose present in the solution. To detect lower levels of biomarkers, Kim et al. proposed a flow-enhanced amperometric detection method, integrating it into a centrifugal microfluidic platform.¹³³ They employed centrifugal-induced continuous flow during amperometric measurements to amplify the signal, achieving a 17-fold improvement in performance.

Voltammetry is another commonly used chemical detection method. It involves measuring the relationship between electrode potential and current to monitor the redox reactions of electroactive substances in the solution, enabling qualitative or quantitative analysis. Compared to traditional voltammetry, cyclic voltammetry is a transient electrochemical testing method that allows for observing the dynamic behavior and stability of electrochemical reactions by scanning the potential multiple times. Andreasen et al. developed a centrifugal microfluidic system for cyclic voltammetry measurements at different rotational speeds, maintaining electrochemical response and enabling data interpretation and quantification.¹³⁴ A microfluidic sensor chip integrating a centrifugal separation pretreatment unit and a composite nano-sensing membrane was developed (Fig. 6C).¹¹⁰ The separated plasma is automatically transferred through the microchannels on the centrifugal microfluidic chip to the detection zone's integrated electrodes for subsequent differential pulse voltammetry detection. During the differential pulse voltammetry process, variations in potential lead to changes in charge transfer between VEGF165 and antibodies, generating signal output. This method achieved a detection limit of 0.67 pg mL⁻¹ for VEGF165 in whole blood.

2.4.4 Other detection strategies. Colorimetric, fluorescent, and electrochemical detection methods are commonly utilized for signal readout on centrifugal microfluidics. In addition to these techniques, other approaches such as surface plasmon resonance (SPR), surface-enhanced Raman spectroscopy (SERS), and distance-based detection have also been employed.^135,136 In particular, distance-based detection techniques involve measuring the distance of a specific substance within a designated channel to obtain detection information.^137,138 This method can be implemented directly within the microfluidic environment without the need for additional chromogenic reagents or labels, thereby reducing dependence on external equipment. Furthermore, this direct data acquisition approach minimizes potential deviations or background interference introduced by reagents, thereby improving the accuracy of the data. For example, Zhu et al. developed a device for rapid visual detection of bacterial growth, which includes a macroscopic bacterial culture chamber for visual observation and a microchannel for bacterial growth detection (Fig. 6D).¹¹¹ After 3–5 hours of incubation, bacteria are concentrated into visible microbar by a single-step centrifugation. This effective enrichment allows for direct comparison of bacterial accumulation under different drug concentrations. Compared to bacteria, viruses are much smaller in size and cannot be cultured using conventional methods. Our team proposed a novel platform based on a manual centrifuge micro-pipette tip.¹³⁹ In the absence of the target antigen, red nanoparticles remain in a free state. When the target SARS-CoV-2 virus antigen is present, it triggers the aggregation of the free red nanoparticles into larger complexes. These complexes accumulate in a narrow channel, forming a “virus bar”. The length of this bar correlates directly with the virus concentration, allowing for visual quantification of SARS-CoV-2 antigen without the need for detection equipment.

2.4.5 Comparison of signal detection methods. On centrifugal microfluidic platforms, the four common signal output methods—colorimetry,¹⁰⁸ fluorescence,¹⁰⁹ electrochemistry,¹¹⁰ and distance¹¹¹—each offer unique benefits and drawbacks. Colorimetry is simple, cost-effective, and easy to operate, making it ideal for preliminary screening or samples with significant color changes, but it has lower sensitivity and is prone to background interference, limiting its use for transparent or low-concentration samples.^95,140 Fluorescence provides higher sensitivity and selectivity by measuring emitted light from excited fluorescent molecules, making it suitable for low-concentration samples, though it requires complex optical systems and is sensitive to background noise and light source instability.¹⁴¹ Electrochemistry, which monitors changes in current, voltage, or conductivity, offers high sensitivity and is cost-effective, ideal for detecting specific chemical reactions, but it can be affected by electrode fouling and lower selectivity in complex samples.^131,142 Distance measurement, which directly assesses liquid levels or object positioning, eliminates the need for external reagents or labels, reducing equipment dependence and minimizing background interference, improving data accuracy and reliability.¹³⁷ However, this method may have lower sensitivity compared to others, limiting its applicability to larger-volume samples. Additionally, some distance-based signals may require amplification to ensure accurate readings, further complicating its use in precise quantification, especially for small-volume or highly sensitive measurements.¹³⁹

3 Applications on centrifugal microfluidic platforms

Centrifugal microfluidics employs straightforward rotational forces to accurately manipulate fluid movement within microscale structures. With its integration and automation, it is an ideal choice for rapid diagnostic systems, especially POCT.^143,144 Through continuous development and iterations, the centrifugal microfluidic platform has evolved into a fully integrated, automated tool for immunoassays, nucleic acid testing, and antimicrobial susceptibility testing, among others (Fig. 7).¹⁴⁵

Fig. 7 Flow chart of centrifugal microfluidic technology and its application. Blue, green, yellow and red represent immunoassay (IA), nucleic acid testing (NAT), antimicrobial susceptibility testing (AST) and others (OT), respectively.

3.1 Immunoassays on centrifugal microfluidic platforms

Immunoassays harness the selective binding between antibodies and antigens, proving essential in diagnosing diseases and tailoring personalized medical treatments.^146–148 Automated immunoassays on centrifugal microfluidic platforms streamline complex biochemical processes into compact, portable devices, which represent a significant advancement in diagnostic technology. Immunoassays are categorized into homogeneous and heterogeneous methods, distinguished by whether the separation of reaction components occurs between a solid and a liquid phase.¹³

3.1.1 Homogeneous immunoassays. In homogeneous immunoassays, all reactants interact directly within a single solution, thus eliminating the need for any separation or washing steps.¹⁴⁹ The direct interaction within the solution allows for continuous monitoring and immediate detection of changes, such as shifts in fluorescence or enzymatic activity, making it particularly effective for rapid testing and real-time analysis in clinical diagnostics and research settings. The most common type of homogeneous immunoassay operates on the principle of energy transfer, where two labeled molecules bind in the presence of an analyte, facilitating an energy transfer that results in signal emission.¹⁵⁰ To integrate analysis systems, Shi et al. developed an all-in-one centrifugal system for early detection of pepsinogen (Fig. 8A), which automatically separates whole blood and perform Alphalisa immunoassays within 12 min.¹⁵¹ For micromolecules, Hatch et al. proposed diffusion immunoassay in a T-sensor by measuring the distribution of a labeled probe molecule after it diffuses for a short time from one region into another region containing antigen-specific antibodies. However, since diffusion transport measurements were conducted under non-equilibrium conditions, ensuring accurate results requires the use of high-precision instruments.¹⁵² To further visualize the signal output and improve the universality, immuno-turbidimetry was proposed, and the content of the compound to be tested can be analyzed by detecting the immunocomplexes suspended in the buffer by a spectrophotometer or naked eye.¹⁵³ Quantifying turbidity signals through naked-eye observation is challenging and can be highly subjective. Based on this, our group proposed a hand-powered centrifugal micro-pipette tip strategy for all-in-one homogeneous immunoassays (Fig. 8B).¹³⁹ After incubation and centrifugation, nano-beads and the target SARS-CoV-2 virus antigen form a complex visible to the naked eye at the bottom of tip, enabling sensitive (1 ng mL⁻¹ for SARS-CoV-2 virus) and reliable quantification within 25 minutes.

Fig. 8 Centrifugal microfluidics for immunoassay applications. (A) Schematic diagram of the centrifugal-Alphalisa system for immune detection (reprinted with permission from ref. 151). (B) Working principle of the hand-powered centrifugal micropipette-tip for the detection of SARS-CoV-2 (reprinted with permission from ref. 139). (C) Schematic and picture of surface plasmon resonance for non-target immunoassay (reprinted with permission from ref. 154). (D) Principle of the fully integrated and high-throughput microfluidic system (reprinted with permission from ref. 155). (E) Detection procedure of the ECL-M POCT device for biomolecules (reprinted with permission from ref. 156). (F) Operation of enhanced centrifugation-assisted lateral flow immunoassay (reprinted with permission from ref. 108).

3.1.2 Heterogeneous immunoassays. In heterogeneous immunoassays, antibodies or antigens anchored to a solid support capture target molecules from the solution. Subsequent washing steps remove any unbound substances, facilitating a quantitative analysis with high specificity and sensitivity.¹⁵⁷ The simplest form of heterogeneous immunoassay is the direct immunoassay, which utilizes antibodies to detect the target analyte directly, with labeled antibodies producing the final analytical result. It involves just one interaction between the antigen and antibody, streamlining the procedure, yet it demands the manual fixation of analytes. To reduce the complexity of antibody labeling and incubation, researchers have explored label-free immunoassays, which simplify the analysis process. For example, Miyazaki et al. developed a centrifugal microfluidic system with the centrifuge-pneumatic siphon valve for sample-to-answer automated SPR detection of IgG (Fig. 8C).¹⁵⁴ It allowed fluidic process integration and parallelization of plasma extraction, metering and aliquoting with selectable incubation times and final SPR detection of five immunoassays within 1 h. Although direct immunoassays are straightforward to perform, they often suffer from low sensitivity and are prone to high background levels that can lead to false positive results. Indirect immunoassays employ unlabeled primary antibodies to capture targets, followed by labeled secondary antibodies that amplify the signal, thus increasing both the sensitivity and specificity of the testing. Enzyme-linked immunosorbent assay (ELISA), the most common form, detects and quantifies analytes through secondary antibodies labeled with enzymes.¹⁵⁸ It is evident that ELISA involves multiple steps, including at least two antigen–antibody reactions and a washing process. Automated ELISA on centrifugal microfluidic platforms has greatly improved the efficiency and accuracy of the testing. For high throughput and multi-target analysis, our group demonstrated an integrated centrifugal microfluidic platform with dual siphon valves and air-pressure balanced structure for multi-target ELISA (Fig. 8D).¹⁵⁵ It conducted high-precision ELISA on a single chip, simultaneously analyzing up to 17 samples or targets, uses only 2% of the reagents compared to conventional ELISA, and employs microbubble-accelerated reactions to halve the assay time. In addition to active mixing, increasing the specific surface area, commonly through the use of porous materials to immobilize antibodies, is also employed to accelerate antigen–antibody reactions and thus enhance sensitivity. Extending this approach, the transition from static to dynamic ELISA leverages materials like magnetic beads to facilitate rapid and sensitive assays by dynamically manipulating reaction conditions.⁶⁸

Beyond optimizing antibody immobilization, diversifying the types of signal outputs, such as different labels, enhances analytical performance. Specifically, fluorescent immunoassays employ fluorescent tags instead of enzymatic chromogenic reactions for higher sensitivity along with quicker data acquisition.¹⁵⁹ In contrast to ELISA, fluorescent immunoassays necessitate specialized equipment to excite fluorescence and measure the resulting emitted fluorescence. Kong's group established a portable centrifugal microfluidic system integrating a homemade fluorescence detection analyzer for sensitive, rapid, multiple, and onsite immunoassays.¹⁶⁰ Chemiluminescence immunoassays are another step up, with photon counting-based determination techniques, which allow for extremely sensitive, rapid quantification of a high dynamic range of analytes. Delgado et al. presented a wirelessly electrified centrifugal microfluidic platform for fully automated chemiluminescence detection for the first time.¹⁶¹ On-line control makes a continuous measurement while the disc is spinning. For low abundance protein detection, Kong's group further described an alternating current electroosmotic flow-based ultrasensitive electrochemiluminescence centrifugal microfluidic system.¹⁶² It achieved an LOD of 2 fg mL⁻¹ for cTnI in 5 min, demonstrating 100% sensitivity and 98% specificity. In addition, they developed a dual-mode multiclassification centrifugal platform with an electrochemiluminescence sensor and a field-effect transistor sensor to minimize occasional inaccuracy in resource-limited and rapid POCT settings (Fig. 8E).¹⁵⁶

LFIA presents a new solution for rapid on-site detection and portability.^163,164 It utilizes antibody–antigen binding and visual labels like colloidal gold for rapid detection, structured into test strips comprising sample pads, conjugate pads, and chromatographic membranes for streamlined analysis. LFIA is popular due to its low cost and simplicity, but has limited sensitivity and weak reagent handling capacity. In view of this, Xu et al. established an enhanced centrifugal-assisted LFIA for rapidly detecting human prostate specific antigen in whole blood (Fig. 8F).¹⁰⁸ Incorporating a nitrocellulose membrane into a centrifugal microfluidic system facilitated fully automated procedures, including sample preparation, active lateral flow actuation, washing, and signal amplification, achieving a detection limit of 0.028 ng mL⁻¹, which represents a 21.4-fold improvement over traditional LFIA.

3.1.3 Digital immunoassays on centrifugal microfluidic platforms. Traditional centrifugal microfluidic immunoassays offer notable enhancements in automation, repeatability, and efficiency, concurrently reducing reagent consumption and labor costs. However, despite these advancements, they encounter limitations regarding detection sensitivity and quantitative accuracy, particularly when confronting low-abundance proteins.^165,166 The advent of digital immunoassays on centrifugal microfluidic platforms, propelled by advancements in biotechnology and nanotechnology, addressed these challenges. Digital immunoassays achieve heightened sensitivity and specificity in detecting antigen–antibody interactions by cutting-edge methodologies like single molecule detection and microfluidics.^167,168 At the core of this approach lies the conversion of conventional immunoassay signals into quantifiable digital signals, wherein each signal denotes a distinct molecular occurrence. This pivotal transformation not only significantly enhances the accuracy and sensitivity but also heralds a transformative advancement in the realm of immunoassays. In 2010, the Walt group first introduced the concept of digital enzyme-linked immunosorbent assay technology for protein ultrasensitive detection via loading the microsphere into fL-volume microwells.¹⁶⁹ The fluorescence signal, catalyzed by enzyme-conjugated streptavidin-β-galactosidase, served as the final output signal.^169–171 In addition to this digital analysis based on physical compartments, droplet based digital immunoassays were exploited to further improve the measurement capacity, which have no limit to the number of chambers.^172–174

Droplet generation through microfluidic technology predominantly relies on channel architecture and the shear and inertia forces induced by fluid flow. Different channel structures lead to various droplet formation mechanisms, including T-junction channels, co-flow channels, flow-focusing channels, and step emulsification.^166,175 Notably, step emulsification simplifies system design and operation, eliminating the need for multi-phase continuous sampling.^176,177 This attribute amplifies its adaptability, rendering it applicable across a wide range of scenarios. Centrifugal step emulsification drives the fluid by centrifugal force, resulting in the creation of a stable liquid film within the microchannel and the dispersion of uniform-sized droplets. Takeuchia's group proposed a centrifuge-powered step emulsification device housed in a microtube for efficient generation of monodisperse picoliter droplets (Fig. 9A).¹²⁶ This setup enabled both cell-free protein synthesis reactions within monodisperse picoliter droplets and the production of glucose-responsive hydrogel microbeads with dimensions comparable to cells. In efforts to enhance system integration, Clime et al. devised a pneumatically-actuated microfluidic platform with buoyancy-driven step emulsification (Fig. 9B).¹⁷⁸ It regulated droplet size by individually tuning the injection speed at the step junction and the buoyancy force on the discretized phase. Moreover, Paust's group introduced dual-volume centrifugal step emulsification for sample metering, facilitating the automated generation of two-droplet populations on a single chip.¹⁷⁹ This advancement resulted in a dynamic range increase of two orders of magnitude compared to previous single-volume platforms.

Fig. 9 Digital immunoassays on centrifugal microfluidic platforms. (A) Concept and operational principle of the centrifuge-based step emulsification device (reprinted with permission from ref. 126). (i) Components of the device. (ii) Schematic of droplet generation by step emulsification. (B) Formation of water-in-oil emulsions using a centrifugal microfluidic platform with pneumatic actuation (reprinted with permission from ref. 178). (i and ii) Schematic illustration of the microfluidic circuit. (iii) Photograph of the centrifugal microfluidic platform. (iv) Close-up view of the emulsification chamber and the microfluidic circuit used for droplet generation. (C) Overview of the centrifugal droplet digital protein detection workflow (reprinted with permission from ref. 180). (i) Sample dilution. (ii) Immunocomplex formation. (iii) High parallel droplet generation with a centrifuge. (iv) PCR cycles yield fluorescent ‘positive’ droplets containing a single antigen. (v) Data analysis and report. (D) Single-molecule protein detection by centrifugal droplet immuno-PCR and magnetic nanoparticles (reprinted with permission from ref. 181). (i) Formation of the immunocomplex. (ii) Separation of targets in microdroplets. (iii) Single molecule counting.

The implementation of a single molecule immunoassay generally necessitates the dilution of samples to extremely low concentrations. Therefore, ensuring the generation of a robust signal is paramount, given the inherent difficulty in capturing single molecule fluorescence or color signals using conventional equipment. Apart from the aforementioned enzyme-catalyzed fluorescent substrate methods, immunoassays coupled with nucleic acid amplification techniques are also frequently employed for signal acquisition.^182,183 This approach leverages the specificity of immunoassays and the sensitivity of nucleic acid amplification, enabling not only signal amplification for stable results but also efficient and accurate target analysis. For example, Wang's group developed a centrifugal droplet digital protein detection technology, which integrated highly parallel centrifugal droplet generation with a wash-free digital immuno-PCR assay, achieving a femtomolar LOD for target proteins in sub-microliters of plasma (Fig. 9C).¹⁸⁰ They incorporated 16 identical droplet generators into a single microchannel array chip, which can be easily assembled into a centrifugal device. A bead-free digital immuno-PCR assay with proximity DNA ligation was designed simultaneously, delivering ultra-high sensitivity and accuracy without the need for multistep washing, as demonstrated by a 0.0128 pg mL⁻¹ LOD for recombinant interleukins (IL-3 and IL-6). Similar to the step emulsification principle, the He group innovated a pipet tip-based microdroplet generator with glass capillary for single-molecule protein analysis (Fig. 9D).¹⁸¹ Sandwich type immunocomplexes were formed off-chip, and PCR amplification with DNA-labeled antibodies within microdroplets yielded the final amplified signal. It achieved ultrasensitive detection of α-synuclein with an LOD of ∼50 aM in buffer and ∼170 aM in serum.

3.2 Nucleic acid analysis on centrifugal microfluidic platforms

Nucleic acid testing, which involves the detection and identification of specific DNA or RNA sequences, is crucial for applications across medical diagnostics, forensic science, pathogen detection, and biological research.^62,184–186 Techniques based on nucleic acid detection enable the precise identification of pathogens and genetic mutations, significantly enhancing the rapid diagnosis of infectious diseases, early detection of genetic conditions, and customization of treatment strategies.¹⁸⁷

3.2.1 Nucleic acid extraction on centrifugal microfluidic platforms. Nucleic acid extraction is the process of separating and purifying DNA or RNA from biological samples, a crucial step that precedes sample amplification.¹⁸⁸ It can ensure the purity of the test samples and avoid impurities from affecting downstream experiments, which directly affect the test results. The nucleic acid extraction process can usually be divided into two steps: lysis and purification.

The methods for disrupting and lysing samples can be categorized into two types:²³ physical methods and biochemical methods. Physical methods for lysis, such as manual grinding, laser, and mechanical disruption, often require specialized equipment, whereas biochemical methods use chemical detergents or enzymes to disrupt membrane structures. Generally speaking, there is less residue in the extract left by the physical method. However, it requires peripheral equipment, such as an ultrasonic meter for mechanical cracking,¹⁸⁹ a peripheral laser system for laser cracking,¹⁹⁰ and a high-voltage power supply for electroporation cracking,¹⁹¹ which is usually completed outside the chip. As for biochemical methods, they are easier to implement within the chip for automated analysis. Despite this, the chemical reagents involved may leave residues that could interfere with subsequent amplification analysis, and their efficiency is somewhat lower than that of physical methods. Overall, researchers are inclined to use biochemical methods and are actively investigating ways to enhance extraction efficiency while eliminating residues during subsequent extraction stages.

To ensure the effective extraction and purification of nucleic acids from lysed samples, it is crucial to eliminate any residual or sample-associated inhibitors that could impact subsequent reactions.¹⁹² Centrifugal precipitation is a traditional method for purification, mainly through the removal of cell debris by centrifugation, followed by the use of organic solvents and solutions to further centrifuge and remove proteins, lipids, and detergents from the solution. Although these methods typically yield highly pure and stable nucleic acid samples, their complex purification processes are challenging to implement on-chip. Additionally, residual reagents such as phenol and chloroform can affect subsequent applications, necessitating the consideration of more suitable methods for chip integration.

Centrifugal microfluidic chips commonly employ special materials that bind with nucleic acids, such as silicon-based and magnetic beads, to simplify processes and enhance efficiency. The functionalized magnetic beads can selectively adsorb nucleic acids and achieve rapid purification through magnetism.¹⁹³ The reversible binding of nucleic acids to magnetic beads can be controlled by adjusting the pH or salt concentration of the solution. The extraction of nucleic acids by magnetic beads is implemented through two primary methodologies: dynamic and static bead manipulation. In the dynamic approach, magnetic beads are maneuvered through different compartments by external magnetic and centrifugal forces. For instance, Hatami et al. designed a fully automated centrifugal microfluidic platform for extracting fetal cell-free DNA from whole blood (Fig. 10A), utilizing three external magnets and varying flow rates to achieve the chamber-to-chamber displacement of magnetic silica beads.¹⁹⁴ Conversely, the static method involves anchoring magnetic beads within a specific segment of the device while sequentially introducing the sample fluid across them to enable reaction completion. Turiello et al. designed a centrifugal disc with laser valves for extracting RNA from SARS-CoV-2 (Fig. 10B), where nano-magnetic beads first bind to viral molecules, and after viral envelope lysis and RNA release, the nucleic acids are retained for subsequent detection.¹⁹⁵ Silicon, with its biocompatibility and stability, is also used for nucleic acid extraction, balancing purification efficacy with cost control.¹⁹⁶ Nucleic acids can be adsorbed or eluted from silicon by altering pH or salt concentration, eliminating the need for complex on-line magnetic control technologies and thereby reducing instrument complexity. Various forms of silicon-based media are employed in nucleic acid extraction. Park et al. showcased an integrated centrifugal microfluidic platform with siphon and capillary valves for nucleic acid testing.¹⁹⁷ Additionally, Xiao et al. designed an on-chip silicon membrane module with Coriolis force-mediated fluid switching for nucleic acid extraction (Fig. 10C).⁵⁶ At the same time, researchers are investigating novel materials for nucleic acid extraction in an effort to enhance the efficiency of the process. Chitosan, with its biocompatibility, biodegradability and porosity, has been increasingly recognized as promising for nucleic acid extraction. Wang et al. directly modified the chitosan channel in the flow channel within centrifugal microfluidic platforms for nucleic acid purification (Fig. 10D).¹⁹⁸ Chitosan can adsorb nucleic acids through electrostatic adsorption and hydrogen bonding in acidic solutions, and the adsorbed nucleic acids can be released in alkaline solutions. It achieved nucleic acid extraction without the introduction of organic reagents, and better avoided potential amplification inhibition.

Fig. 10 Nucleic acid extraction. (A) Schematic diagram of whole blood extraction of cffDNA by a fully automatic centrifugal microfluidic device based on magnetic silica bead replacement (reprinted with permission from ref. 194). (B) Sample preparation sequence unit operation with fixed magnetic beads, including reagent and sample loading, enrichment, waste removal, extraction, and viral RNA elution (reprinted with permission from ref. 195). (C) Schematic and workflow of the fully integrated and automated centrifugal microfluidic chip for point-of-care multiplex molecular diagnostics (reprinted with permission from ref. 56). (D) Workflow of centrifugal microfluidic platform with the PDMS channel modified by chitosan for DNA extraction and purification (reprinted with permission from ref. 198).

3.2.2 Nucleic acid amplification. Nucleic acids are typically found at low concentrations in real samples, making them difficult to detect. To overcome this, amplification of nucleic acids is employed to increase the detectability of minute quantities and enhance overall detection efficiency.¹⁹⁹
3.2.2.1 Polymerase chain reaction amplification. Polymerase chain reaction (PCR) represents the classic amplification technique and is recognized as the gold standard for nucleic acid testing.^200,201 PCR proceeds through a sequence of cycles encompassing denaturation, annealing, and extension, using heat-stable DNA polymerases to rapidly and specifically replicate target nucleic acid fragments. PCR-based microfluidic chips can be categorized into chamber-based fixed PCR²⁰² and continuous flow PCR (CF-PCR).²⁰³ In chamber-based systems, reagents remain in fixed chambers that cycle through various temperatures to conduct the PCR process. For example, Stumpf et al. developed a microfluidic chip equipped with comprehensive reagent pre-storage that sequentially released a high-wettability extraction buffer for conducting real-time reverse transcription PCR.¹⁸⁸ For further application, Guo et al. described a pneumatic centrifugal microfluidic platform for PCR amplification to construct high-quality DNA libraries (Fig. 11A).²⁰⁴ Air pressure applied to one or more pneumatic lines during centrifugation was used to trigger various fluidic actions inside the microfluidic devices. In CF-PCR, reagents continuously flow through areas with different temperatures to complete the amplification. Compared to the original chamber-based fixed PCR, CF-PCR addresses the issue of discontinuous and time-consuming processes caused by temperature fluctuations in traditional PCR. CF-PCR speeded up DNA amplification by circulating reagents through three pre-heated zones, employing designs like serpentine, spiral, or short straight channels to seamlessly transition between different reaction temperatures, thus significantly shortening the analysis time.²⁰⁵ Jung et al. proposed a rotating PCR gene analyzer, which integrates characteristics of both chamber-based and continuous flow PCR for reverse transcription PCR.²⁰⁶ The heating system below the chip is divided into three modules responsible for denaturation, annealing, and extension, respectively, allowing it to amplify the target gene within just 25.5 minutes.

Fig. 11 Nucleic acid amplification. (A) Centrifugal microfluidic chip scheme for preparing high-quality DNA libraries by fixing DNA with magnetic beads for secondary purification after PCR (reprinted with permission from ref. 204). (B) Solution for fast and fully automatic detection of bacterial pathogens by high-sensitivity nested PCR on a centrifugal microfluidic platform (reprinted with permission from ref. 207). (C) Schematic view of the centrifugal microfluidic platform for rapid, monoplex and colorimetric detection of foodborne pathogens by LAMP (reprinted with permission from ref. 78). (D) Flow diagram and details of the LAMP-microfluidic assay (reprinted with permission from ref. 208). (E) Schematic illustration of the centrifugal direct-RPA microfluidic platform (reprinted with permission from ref. 209). (F) Combining the immunomagnetic separation technology of enriched target bacteria and the rapid detection scheme of RAA bacteria (reprinted with permission from ref. 210).

Nested PCR, as a technique to improve the sensitivity and specificity of PCR, uses two pairs of PCR primers to amplify complete fragments.²¹¹ The first pair of primers amplifies a fragment similarly to standard PCR, while the second pair, known as nested primers, is positioned within the first PCR amplified fragment, resulting in a shorter fragment in the second PCR. Positioned closer to the center of the target sequence, the second round of primers reduces the likelihood of non-specific binding, thereby enhancing the accuracy of the entire reaction.²¹² For example, Czilwik et al. presented a highly-sensitive LabDisk-Automated-Assay for a nested PCR-based detection of multiple pathogens (Fig. 11B).²⁰⁷

3.2.3 Isothermal amplification. Although PCR has the advantages of high sensitivity and accurate quantification, thermal cycling makes the integration process difficult and costly.²¹³ In order to avoid the thermal circulation system of PCR, further reduce the complexity of the nucleic acid amplification system, and improve the amplification efficiency through automated detection methods, isothermal amplification technology came into being.¹⁹⁹

Among the currently available isothermal nucleic acid amplification methods, loop-mediated isothermal amplification (LAMP) is one of the most widely used.²¹⁴ Its principle involves the use of chain-displacement Bst DNA polymerase and 4–6 primers to facilitate self-cycling at temperatures ranging from 60–65 °C.²¹⁵ The “loop” primers in LAMP create self-amplifying stem-loop structures that enable the replication of their sequences in each cycle, continuously linking with previous amplicons to produce varying lengths of tandem repeat products. In LAMP systems, colorimetric detection is the most commonly used with its easy use. Oh et al. proposed a centrifugal LAMP microdevice with zigzag-shaped microchannels for sequential aliquoting of the LAMP components.²¹⁶ It enabled multiplex foodborne pathogen identification with Eriochrome Black T-mediated colorimetric detection for LAMP products. Besides, Sayad et al. demonstrated a more integrated centrifugal microfluidic system with pumping, mixing, metering, amplification and detection for calcein-based colorimetric LAMP (Fig. 11C).⁷⁸ Although they integrated the full LAMP amplification process, it lacked extraction steps and is not suitable for real sample analysis. The integration and automation of nucleic acid testing necessitate the combination of nucleic acid extraction with the pre-storage of certain reagents for subsequent amplification. Despite this, colorimetric LAMP is still limited with its application for environmental variables, particularly the pH levels of samples.²¹⁷ Fluorescent LAMP systems offer a more robust alternative to colorimetric detection by using fluorescent dyes that bind to the amplified DNA. For example, Kong's group presented a LAMP-based centrifugal microfluidic platform for rapid genotype detection and diagnosis of seven coronaviruses (Fig. 11D).²⁰⁸ For integrated nucleic acid analysis with extraction and amplification, our group designed an integrated and automated centrifugal microfluidic platform with a pneumatic balance module for sequential release of multiple reagents, a pneumatic centrifugation-assisted module for on-demand solution release, an on-chip silicon membrane module for nucleic acid extraction, a Coriolis force-mediated fluid switching module, and an amplification module for fluorescent LAMP.⁵⁶

LAMP technology excels in efficiency, providing a robust method for the rapid amplification of nucleic acids, which facilitates quick and accurate detection across various scientific and medical contexts.¹⁹⁹ However, to achieve similar nucleic acid amplification at lower temperatures and with simpler equipment, we can transition to recombinase polymerase amplification (RPA) technology, which allows for effective amplification at ambient temperatures.^218,219 In the system, two opposite primers are combined with recombinase to initiate DNA synthesis, and the detection speed is faster than PCR and LAMP.¹⁸⁵ According to the type of recombinase used, two types can be distinguished, namely recombinase polymerase amplification and recombinase-mediated amplification (RAA).²²⁰ The recombinase of RPA is derived from T4 bacteriophages, while the recombinase of RAA is derived from bacteria or fungi. Choi et al. developed a centrifugal direct RPA microdevice for multiplex and real-time identification of food poisoning bacteria (Fig. 11E).²⁰⁹ The bacterial cells spiked in milk were lysed by the direct PCR buffer and the target genes were amplified by the RPA reagents without the sample pretreatment step. Although it successfully achieved a detection sensitivity of 4 cells per 3.2 μL of milk within 30 min, it currently appears to be suitable only for mock samples, not real ones. Kim et al. presented a centrifugal microfluidic system with DNA extraction, isothermal RPA, and detection for rapid molecular diagnostic analysis.²²¹ However, sample enrichment was achieved off-chip by magnetic bead-coupled antibodies, and only lysis rather than extraction and purification was demonstrated on the on-chip. Wang et al. proposed an automatic centrifugal system for sample-in-result-out quantification of foodborne pathogens, which enhanced amplification efficiency and its applicability to a wide range of sample pathogens (Fig. 11F).²¹⁰

3.2.4 CRISPR-based nucleic acid detection. Isothermal amplification excels in situations where rapid and efficient amplification is needed at a consistent temperature. While this is effective for many applications, it still involves the amplification of nucleic acids, which can be time-consuming and technically demanding in some contexts. Moving to amplification-free detection methods, these eliminate the need for nucleic acid amplification, simplifying the workflow and reducing the potential for errors related to complex amplification steps.^199,222 Building on this progression, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based techniques capitalize on the specific advantages of amplification-free methods, adding unparalleled specificity and versatility. By specifically designed single-guide RNAs (sgRNAs) to precisely recognize target DNA or RNA sequences for accurate cleavage or modification, CRISPR systems enable precise genetic analysis, making them ideal for applications where accuracy and speed are paramount.^223,224 Cas proteins such as Cas9, Cas12a, and Cas13a have been widely applied in nucleic acid analysis and have demonstrated their feasibility as diagnostic tools.^225,226 Notably, standalone CRISPR detection was not as effective as combined isothermal amplification since amplification can enhance the sensitivity of detection. CRISPR combined with isothermal amplification not only mitigates the risk of false positives associated with amplification but also decreases the time of amplification. Nevertheless, combining LAMP with CRISPR also presents challenges, such as differing reaction temperature conditions and competitive interactions between the two methods. Yin et al. addressed these issues by integrating a centrifugal microfluidic chip, a dual-temperature heating module, and a portable device based on a smartphone application within an RT/LAMP-CRISPR/Cas12a system (Fig. 12A), resolving temperature discrepancies and competitive issues while ensuring a detection speed of just 45 minutes.²²⁷ By dividing the amplification and detection processes into separate chambers, this approach necessitates more sophisticated temperature control and fluid management systems. Compared to LAMP, RPA technologies demonstrate superior temperature adaptability and their integration with CRISPR-Cas systems showcases a high level of complementary functionality.²²⁸ The similar reaction temperatures of RPA and CRISPR allow them to be conducted together in one chamber. For example, Zong et al. developed a fully integrated and automated centrifugal microfluidic chip with pretreatment, extraction, and detection for genotyping HBV from whole blood (Fig. 12B).²²⁹ This one-pot method reduces the need for intricate temperature control and simplifies the fluid handling process. With more integrated signal output, Wang's group proposed a portable centrifugal microfluidic testing system for “sample-to-answer” RPA and CRISPR detection of infectious pathogens from plasma samples.²³⁰

Fig. 12 Nucleic acid analysis applications based on CRISPR technology. (A) Schematic illustration of the SEDphone system workflow based on CRISPR/Cas12a and RT/LAMP technology (reprinted with permission from ref. 227). (B) Automated workflow on the centrifugal microfluidic chip with the RPA-T7-Cas13a system for genotyping of hepatitis B virus from whole blood (reprinted with permission from ref. 229).

3.3 Antimicrobial susceptibility testing on centrifugal microfluidic platforms

In recent decades, excessive use of broad-spectrum antibiotics has caused a pervasive increase in antimicrobial resistance, posing a serious global public health threat.^231,232 Hence, accurate microbial identification and rapid resistance monitoring are crucial for tackling this challenge. Antimicrobial susceptibility testing (AST) assesses how pathogenic microorganisms respond to specific antibiotics, which guides clinicians in selecting the appropriate treatment, especially in combating antibiotic-resistant bacteria.^233,234 Traditional AST methods, such as broth dilution and disk diffusion, typically require culturing bacteria for 18 to 24 hours, followed by additional time to assess bacterial growth patterns in the presence of antibiotics.²³⁵ This entire process can take several days—from specimen collection to result interpretation—making it both time-consuming and labor-intensive. The integration of all steps on centrifugal microfluidic chips for AST, encompassing concentration gradient generation, sample distribution and result readout, underscores its benefits in speed, automation and efficient resource management.

To automatically generate gradient antibiotic solutions, Tang et al. present a 3-dimensional multi-layered centrifugal microfluidic platform for the generation of linear concentration gradients via metering chambers (Fig. 13A).²³⁶ It successfully performed AST within 3 hours by determining the minimum inhibitory concentration (MIC) of ampicillin against E. coli. More simplistically, our group developed an electricity-free, portable, and robust handyfuge microfluidic chip for on-site AST with a pH-based colorimetric strategy (Fig. 13B).⁷⁶ Accurate antibiotic concentration gradients within bacterial–antibiotic mixtures could be generated in under 5 minutes, with MIC values for various antibiotics obtainable within 5 hours. The handyfuge-AST system also achieved 100% categorical agreement with the clinical results. In addition to this volume-dependent method, diffusive mixing also fulfills the demand for generating concentration gradients. For example, Pang et al. proposed a controlled-diffusion centrifugal microfluidic platform to generate antibiotic concentration gradients for rapid AST.²³⁷

Fig. 13 Centrifugal microfluidic for AST. (A) Top view of the three layers of the centrifugal microfluidic platforms for AST (reprinted with permission from ref. 236). (B) Flowchart for MIC using handyfuge microfluidics (reprinted with permission from ref. 76). (i) Schematic diagram of the whole operation process of handyfuge-AST for urine samples with the mobile app. (ii) Cloud-based diagnostic support. (C) Schematic of the concentration process using centrifugal force in the microfluidic chip (reprinted with permission from ref. 238). (D) Workflow of rapid bacterial separation and AST from PBCs (reprinted with permission from ref. 239). (E) Dx-FS as a versatile bacterial infection diagnostic platform (reprinted with permission from ref. 240). (i) Operation of a Dx-FS device. (ii) Fluorescently labelled bacterial cells enriched using Dx-FS. (iii) Number of bacterial cells detected. (iv) Colorimetric bacterial cell viability assay performed using Dx-FS. (v) Orange colour intensities from iv.

In general, the samples tested directly above need to reach a relatively high concentration, usually around 10⁸ CFU mL⁻¹ (0.5 McFarland standard). The original sample, especially blood samples, lacked such high concentrations, necessitating extended lengthy culture periods.²⁴¹ Consequently, it is essential to conduct AST for bacterial infections with low concentrations. Centrifugation for sample concentration is a practical approach that effectively reduces detection limits, minimizes unnecessary culture time, and shortens the testing duration.²³ Specifically, Hwang et al. introduced a centrifuge enrichment system with bacterial traps at the end for rapid AST (Fig. 13C).²³⁸ Time-lapse images without any labels were employed for tracking the growth of bacteria by counting numbers. It demonstrated rapid and accurate AST within 3 hours at a bacterial sample concentration of 10³ CFU mL⁻¹. Given the complexity of original bacterial samples, Zhu et al. introduced an integrated chip for direct AST from positive blood cultures without subculture (Fig. 13D).²³⁹ It could extract bacteria directly from PBCs within 10 min and quickly give susceptibility information by percentage of bacterial area in taper tips within 3 h. For easy data readout, they also developed a capillary-based centrifugal indicator for fast and accurate AST by the assessment of packed capillary column height.²⁴² In another work, Cho's group described a diagnostic fidget spinner (Dx-FS) that rapidly concentrated pathogens in 1-mL samples of undiluted urine by more than 100-fold for the on-device colorimetric detection of bacterial load and pathogen identification (Fig. 13E).²⁴⁰ The Dx-FS was a palm-sized device powered by hand that enabled on-site detection of infection visible to the naked eye in 50 min and low-cost electricity-free AST within 120 min.

3.4 Other tests on centrifugal microfluidic platforms

3.4.1 Separation and quantification of biochemical substances. The biochemical substances in samples can usually include two types of biological particles and abiotic substances. The analysis of biological particles is mainly reflected in the extraction, quantification and detection of small molecules such as glucose and creatinine.^131,243 Abiotic substances typically refer to chemical substances that originate from non-living sources, such as ions and inorganic compounds.^134,244

In the separation and extraction of biochemical substances, centrifugal microfluidic platforms naturally excel due to the inherent centrifugal force and Coriolis effects. Platelets, being small and low-density, typically need to undergo high-speed centrifugation in traditional separation processes, which can lead to excessive activation. Moreover, with small sample volumes, traditional methods may struggle to effectively separate platelets, raising the risk of contamination. Through the integration of sequence and tangential flow filtration modules, Cho et al. developed a fully automatic centrifugal microfluidic chip for separation of high-purity platelets (Fig. 14A), replacing the precipitation process of the traditional method with filtration-based separation, with milder centrifugation conditions, higher purity, and all operations can be completed within 20 min.²⁴⁵ Additionally, centrifugal microfluidic platforms play a significant role in environmental monitoring, particularly in the detection of environmental contaminants like heavy metal ions in water samples.

Fig. 14 Other tests on centrifugal microfluidic platforms. (A) Configuration of the centrifugal microfluidic platform for pure platelet isolation (reprinted with permission from ref. 245). (B) Schematic diagram of the sorting port (reprinted with permission from ref. 246). (C) Schematic illustrations showing the sorting mechanism with flow rectifier (reprinted with permission from ref. 247). (D) Schematic diagram of centrifugal microfluidics for liver function testing (reprinted with permission from ref. 248).

Traditional detection methods, such as atomic absorption spectroscopy and atomic fluorescence spectroscopy, offer significant advantages in sensitivity and accuracy. However, their reliance on complex equipment and procedures, as well as the stringent requirements for installation environments and maintenance, limit their applicability in field testing. Zhou et al. proposed a centrifugal platform with sampling, mixing, reaction for simultaneous detection of heavy metal ions (Fig. 14B).¹⁰³ The LODs of Pb²⁺, Hg²⁺ and As³⁺ heavy metal ions are 6.16 ppb, 4.97 ppb and 5.24 ppb, respectively. For heterogeneous samples, the aforementioned methods are difficult to apply. Based on the characteristics of heterogeneous samples, the centrifugal microfluidics can convert heterogeneous solutions into droplets encapsulating signal molecules. This approach improves detection throughput and limits of detection. For example, Cai et al. introduced a centrifugal microfluidic chip composed of spiral microchannels.²⁴⁶ Micro-pore arrays are integrated along the sidewalls of the spiral channels, enabling size-based droplet sorting and enrichment under the influence of multiple forces. This provides an efficient, cross-contamination-free sorting method for heterogeneous multi-sample microreactor units.

3.4.2 Cell sorting and detection. The isolation and screening of cells from plasma or whole blood is of great significance in blood analysis, which can reduce background interference and improve detection accuracy.^25,249 This process focuses on effectively isolating key cell types from blood samples, such as red blood cells, immune cells, and cancer cells.^250–252 Each type plays a critical role in diagnosing, monitoring, and treating various diseases. For instance, isolating immune cells helps assess immune response and inflammatory conditions, while detecting and quantifying cancer cells are essential for cancer diagnostics and monitoring therapeutic efficacy.²⁵³ This precise separation technology allows researchers and clinicians to gain deeper insights into disease mechanisms and provide more personalized treatment options for patients. Hence, cell screening not only enhances the quality of subsequent analyses but also significantly improves the accuracy of diagnostic data and the efficiency of clinical decision-making.

Currently, centrifugal microfluidic chips utilize sedimentation for sorting biological particles, separating them based on differences in density. Greater size differences among particles compared to density differences can lead to unstable flow rates within the chip, resulting in low separation purity for particles of varying sizes. Ma et al. proposed a centrifugal microfluidic chip with a flow rectifier (Fig. 14C), which addressed issues of particle sedimentation and unsteady flow in the sample chamber.²⁴⁷ This design significantly improved the recovery rate and separation purity of the target particles and is effective for separating particles of different sizes. Under experimental verification, rare tumor cells were successfully isolated from high-concentration white blood cells, with a recovery rate of 90.4% ± 2.4% and a separation purity of 83.0% ± 3.8%. To enhance sorting specificity and accuracy, Lin et al. designed a microfluidic chip with on-disc labeling for isolating and characterizing rare cells that are difficult to separate using traditional methods (Fig. 14D).²⁴⁸ The feasibility and accuracy of extraction were verified by comparing the short tandem repeat sequence in the genome-wide amplification content with the parent body.

4 Conclusion and outlook

As an important branch of microfluidic technology, centrifugal microfluidic chips are increasingly capturing the interest of the research community. This technology leverages centrifugal force to drive fluid, enabling precise fluid control and processing. It combines the advantages of microfluidic technology with the unique characteristics of centrifugal force, and provides new solutions for the fields of biomedicine, chemical analysis, and environmental monitoring. A key feature of centrifugal microfluidic technology is its ability to conduct complex fluid operations with small rotating devices. Fluids are directed through microfluidic channels on a rotating disc, allowing for precise control over fluid dynamics. This design simplifies instrumentation requirements, reduces experimental costs, and enhances operational convenience. Moreover, the capability of these chips to process multiple samples concurrently significantly boosts experimental throughput and efficiency.

Centrifugal microfluidic chips have demonstrated significant potential in POCT across a variety of fields, particularly enhancing capabilities in immunoassays, nucleic acid testing, and antimicrobial susceptibility. These chips integrate seamlessly with POCT, streamlining complex diagnostic processes into simpler, faster, and more cost-effective procedures. By consolidating multiple testing functions onto a single platform, these chips enable rapid, on-site diagnostic results, which is critical in situations requiring quick decision-making, such as clinical settings, field testing, and emergency situations. This synergy between centrifugal microfluidic chips and POCT represents a transformative advancement in making sophisticated diagnostic tools more accessible and practical for widespread use. However, its development still faces some challenges. First of all, the manufacturing process of centrifugal microfluidic chips requires high-precision process technology to ensure the accuracy and consistency of the microfluidic channels in the chip. Secondly, due to the dependence on centrifugal force, the rotation control of the chip requires precise speed and time adjustment, which requires higher requirements for the control system. In addition, most centrifugal microfluidic chips are still single-use at present, which may increase the cost and environmental burden of long-term use.

It is worth noting that microfluidics, as an engineering technology, does not inherently possess detection capabilities. Therefore, future advancements will require the integration of additional technologies with centrifugal microfluidic systems to fully unlock the potential of these platforms. Currently, centrifugal microfluidic platforms integrate analytical techniques such as PCR, LAMP, ELISA, and LFIA, enabling handheld devices to conduct pathogen nucleic acid or protein detection for infection sources like human papillomavirus and monkeypox virus. However, most microfluidic technologies remain limited to laboratory environments, leaving significant untapped potential for broader applications. As such, future research in microfluidics may concentrate on the following areas. First, the design and manufacturing processes of microfluidic platforms need to be further improved to reduce costs and enhance reusability.²⁰ For example, advanced fabrication techniques such as 3D printing facilitate the production of microfluidic systems, while cost-effective centrifugal platforms like fingertip gyroscopes or hand-crank fans can also be explored for more accessible solutions.^10,254 This would enable faster platform construction at a lower cost. Second, there is a need to develop more advanced control systems that can handle complex fluid manipulations and provide finer experimental precision.^255,256 Increased integration of centrifugal microfluidic platforms could allow for more sophisticated fluid handling capabilities. Third, while most current microfluidic systems are still limited to laboratory environments, expanding their applications is essential. As the healthcare landscape shifts toward personalized medicine and convenient diagnostics, microfluidic platforms must be further miniaturized for home self-testing and emergency use, especially in resource-limited settings. Fourth, increasing the integration and throughput of microfluidic platforms will be crucial, allowing for simultaneous detection of multiple biomarkers such as nucleic acids, proteins, and liposomes. In the future, a single chip could potentially perform multi-parameter analysis and biomarker identification, improving diagnostic capabilities. Fifth, it is essential to foster interdisciplinary collaborations that integrate microfluidic chips with cutting-edge technologies such as artificial intelligence (AI) aiming to create intelligent and automated experimental platforms.^257,258 Such integrations would enable AI to automatically analyze results from microfluidic tests and provide diagnostic reports, helping individuals make informed decisions about self-treatment. Moreover, AI integration can improve diagnostics in resource-poor areas, protect privacy through at-home testing, and enhance healthcare accessibility. During the COVID-19 pandemic, microfluidic devices represented by centrifugal microfluidic platforms demonstrated their capability and advantages in rapid testing, and they hold great promise for future applications, especially in pathogen detection. As technology continues to advance, centrifugal microfluidics is poised to play a greater role in future scientific research and practical applications.

Data availability

No primary research results, software or code have been included and no new data were generated or analysed as part of this review.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

We gratefully acknowledge the National Key Research and Development Program of China (2021YFA1101500, 2023YFF1205002 and 2024YFF1207300), the National Natural Science Foundation of China (22074047, 32171248, 12102142 and 12472319) and the Fundamental Research Funds for Central Universities, HUST (2024107). We also thank to BioRender (http://www.biorender.com/) for providing some of the graphic elements used in this study.

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Footnote

† These authors contributed equally to this work.

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