The whole blood sample was collected from the Jimei University Medical Center and approved by the Jimei University Science and Technology Ethics Committee. The experimental procedure complies with GB 19,489 - 2008 "General Requirements for Laboratory Biosafety". Based on the principle of antigen-antibody immune recognition, blood samples from several volunteers were taken and reacted with anti-A/anti-B monoclonal antibody reagents (Beijing Jinhao Pharmaceutical) at room temperature (25 ± 1 ℃) for 5 min. The agglutination status of red blood cells was observed using an optical microscope (Beijing Senico, SN-LV2000), and blood type interpretation was performed by the "National Clinical Laboratory Operating Procedures" (China).
Directed fluid driving in microfluidic chips is typically achieved using valved micropumps, which induce deformation of the pump membrane by applying periodic pressure, thereby precisely controlling the sequential opening and closing of unidirectional valve pairs. A common method is direct finger pressing, but it suffers from significant errors. Alternatively, gas pressure conduction is used; however, operator variability can lead to fluctuations in fluid delivery, making it difficult to ensure fluid flow precision.
In response to this issue, this study further developed a novel finger-actuated quantitative drive module (Fig. 2(b)). The core mechanism is to achieve precise directional transport of quantitative fluid along a predetermined flow channel by circulating the following operating procedures: (1) initial positioning stage: adjust the support components to be parallel to the surface of the chips integrated valve micropump, ensuring that the driving contacts are accurately aligned with the center of the micropump chamber; (Fig. S3) (2) Pressure driving stage: By applying force with fingers to make the pressing part contact the supporting part, the driving contact presses the micropump membrane to produce downward deformation, compresses the chamber volume and increases the pressure inside the chamber, thereby opening the outlet check valve and achieving quantitative fluid delivery from the chamber to the flow channel; (3) Reset suction stage: After removing the external force, the spring drives all components to reset. The pump membrane deforms upward due to elastic recovery, and the chamber volume expands to form negative pressure. At this point, the inlet checks valve opens, completing quantitative fluid aspiration from the channel to the chamber.
The innovation of this module is mainly reflected in some aspects: (1) The threaded screwing mechanism between the bolt and the pressing component enables precise control of the pressing depth (The force applied by the finger to the bolt head ensures complete contact between the bottom surface of the pressing component and the top surface of the supporting component). (2) The actuation contact-point guarantees a consistent contact area on the pump membrane, while the pressing position remains fixed. (3) The reset function of the spring ensures consistent pressing depth during each actuation cycle. (4) The modular driving component employs a detachable design, allowing the actuation contact points to be topologically optimized to meet the requirements of high-throughput experiments.
These four methods can transform variations in operator input into controllable target parameters, achieving quantitative fluid drive and significantly enhancing the convenience of measuring this critical parameter. The modular driving component employs a detachable design, allowing the actuation contact points to be topologically optimized to meet the requirements of high-throughput experiments.
In summary, compared to the traditional method of direct finger pressing, this module exhibits significant advantages in output stability, flow control accuracy, and high-throughput compatibility. In addition, the module adopts a compact design, which has the characteristics of simple structure and strong portability, and is particularly suitable for POCT applications of microfluidic chips.
As the power source for microfluidic chip actuation, the key parameters of the finger-pressure quantitative actuation module are critical to its performance. As shown in Fig. 2(b) and Fig. 2(c), the pressing depth (h), pressing time interval, and contact dimensions (contact height d and contact diameter D) significantly influence the actuation performance.
The pressing depth (h) of the finger-actuated quantitative actuation module is defined as the distance between the bottom surface of the pressing component and the top surface of the support component (Fig. 2(c)), representing the maximum deformation displacement of the pump membrane in the vertical direction during each press. To quantify the impact of this parameter on fluid actuation performance, this study conducted 10 cycles of periodic pressing tests under fixed chamber geometry parameters (chip actuation chamber diameter (D) = 6 mm, depth (h) = 0.5 mm, pump membrane thickness (t) = 1.5 mm) using incrementally increasing pressing depths (h = 0.2/0.3/0.5 mm). As shown in Fig. 3(a), the cumulative driven fluid volume is proportional to the number of presses. Additionally, a greater pressing depth corresponds to a larger deformation of the pump membrane, resulting in more fluid being expelled or drawn into the pump chamber per press. Consequently, the results indicate that larger pressing depths correspond to increased fluid volumes driven per actuation cycle.
The pressing time interval (T) of the finger-actuated quantitative drive module is defined as the waiting time between two adjacent pressing operations. To quantify the impact of this parameter on fluid driving performance, in this study, experiments were conducted under fixed chamber geometry parameters (D = 6 mm, h = 0.5 mm, t = 1.5 mm) and a constant pressing depth (h = 0.2 mm) with three replicates (n = 3), using varying pressing time intervals (T = 1 s, 2 s, 3 s, and 4 s). By measuring the fluid volume driven per press, it was observed that the driven fluid volume showed no significant difference for T ≥ 2 s, whereas the driven volume at T = 1 s was significantly lower (Fig. 3(b)). This phenomenon can be attributed to two primary factors: first, the deformation and recovery of the PDMS pump membrane are not instantaneous processes and require a finite duration; second, the opening and closing actions of the one-way valve exhibit a certain degree of hysteresis. Consequently, a pressing time interval of ≥ 2 s is necessary to allow sufficient time for the PDMS membrane to fully deform or recover, thereby ensuring stable quantitative fluid actuation by finger pressure.
Additionally, the driving component, as a critical actuator of the module, has geometric parameters of the driving contact (diameter D and height d) that may influence pump membrane deformation and, consequently, fluid actuation performance. To assess this, two controlled experimental groups were established: a diameter gradient group (D = 1 mm, 2 mm, 3 mm; d = 1 mm) and a height gradient group (D = 1 mm; d = 1 mm, 2 mm). Under the same fixed chamber parameters, the fluid volume driven per actuation cycle was measured for varying driving contact dimensions. The results indicate that larger driving contact diameters are associated with enhanced fluid actuation performance, though the differences are not statistically significant (Fig. 3(c)). In contrast, variations in driving contact height have a negligible effect on the driven fluid volume (Fig. 3(d)). These findings suggest that modifying the geometric parameters of the driving contact is not an effective strategy for regulating micropump performance.
Finger-actuated microfluidic chips are susceptible to individual operational differences, which may lead to decreased flow control accuracy and degraded driving consistency. This study focuses on systematically verifying the impact of individual differences among operators on the consistency of driving performance. Under fixed channel geometry parameters (D = 6 mm, h = 0.5 mm, t = 1.5 mm) and standardized finger-actuated quantitative module configurations, conduct experiments with two groups of different chamber depths (0.2 mm and 0.5 mm). Six volunteers were recruited to perform operational tests using the same actuation module and chip, with pressing depth controlled uniformly. Each volunteer performed three presses, and the fluid volume driven per press was recorded (Fig. 4(a)). The results demonstrate that the fluid volume driven per press was relatively consistent across volunteers, with statistical analysis revealing no significant differences among individuals. This validates the stability and robustness of the actuation module under conditions of individual operator variability.
To verify the fluidic driving control precision when multiple micropumps are actuated simultaneously by a single press, a quantitative analysis of the fluid volume driven in each independent channel was performed. Based on the design of the blood type POCT chip, the driving component in this study was designed with four actuation contacts, enabling parallel driving of four channels. First, a pressing depth of 0.3 mm was applied to the four actuation contacts (labeled 1-4). The volume of fluid driven in each independent channel per press was measured, yielding the results shown in Fig. 4b. Statistical analysis revealed no statistically significant differences in the single-press driven fluid volume corresponding to each actuation contact, indicating that the parallel multi-channel driving scheme achieves a high degree of consistency in fluidic driving performance. Furthermore, Fig. 4(c) visually demonstrates the fluid flow results in the blank channels during six consecutive presses. Although minor bubbles appeared at isolated locations within the channels, they did not compromise the overall fluidic driving effectiveness.
The multi-fluid mixing efficiency of chips directly affects the accuracy of POCT results. This study evaluates the hybrid performance of the system to ensure that the chip's design meets the requirements of blood type detection. Establish a visual evaluation system: Use turquoise blue (RGB: 0, 238, 238) and bright red (RGB: 255,0,0) staining solutions as tracers to quantitatively analyze the mixing process of microchannels. By compressing the finger-actuated module integrated on the chips, the two-color solution is promoted to mix in a serpentine channel, and the mixing degree of the solution in the detection chamber is quantitatively analyzed. As shown in Fig. 5(a-c), the dual color solution (turquoise/bright red) is driven by a valve micropump chamber, diffused and mixed in a serpentine mixing channel, and finally uniformly mixed in the detection chamber. Its color continues to evolve, and its RGB values evolve from an initial bimodal distribution to a unimodal distribution, which well verifies the excellent mixing performance of the chips.
This study is based on the principle of antigen-antibody immune recognition, establishing a chip-based real-time blood type detection method, and capturing agglutination reaction color characteristics through a microscopic imaging system. Inject whole blood samples through injection ports 1 and 3, and inject anti-A and anti-B antibody reagents (Whole blood: reagent = 1:1) into injection ports 2 and 4, respectively. After completing the antigen-antibody reaction through a serpentine mixing channel, obtain visualized results in the detection chamber. The integrated finger-actuated module achieves precise control of fluid volume, ensuring proportional mixing and significantly reducing manual operation errors.
Two known blood types (O and A) volunteer samples were selected for double-blind validation experiments. The pre-experiment used a standard coagulation plate to verify the effectiveness of the reagent: specific agglutination was observed when A-type whole blood reacted with anti-A reagent at room temperature (25 ± 1 ℃) for 5 min, while no agglutination was observed between O-type whole blood and anti-A/B reagent (Fig. 5(d)). This result conforms to the antigen-antibody reaction pattern of the ABO blood group system, confirming the reliability and specificity of the reagents used in the experiment. Chips validation protocol: Take 50 µL of O-type whole blood (divided into 2 × 25 µL) and inject it into injection ports 1 and 3. Inject 25 µL of anti-A/B reagent into injection ports 2 and 4, respectively, to construct a parallel detection system. Set the compression stroke of the finger-actuated module to 0.5 mm. After the reaction solution is transported to the detection chamber, react for 5 min at room temperature (25 ± 1 ℃) and record the agglutination image (the same process and parameters for type A blood detection as above). The detection chamber reaction results captured by the microscopic imaging system are shown in Fig. 5(e), where the O-type sample did not show agglutination reaction (anti-A/B reagent), and the A-type sample exhibited typical agglutination with the anti-A reagent, which aligns perfectly with the results obtained using the traditional test tube method. The accuracy of the experiment was further confirmed through standardized testing procedures. However, due to the unavailability of B blood type volunteers during the testing phase, sample acquisition was limited. Moreover, since B blood type testing was not the primary focus of this study, such tests were not performed.
In future work, the microscopy imaging system used in the experiments could be replaced with a smartphone and a portable smartphone magnifier to observe colorimetric features (Fig. S5).