To manipulate liquid/solid mixed phases in the PTM, we put forward a photo/magnet-combined control strategy, and employed magnetic nanoparticles (MNPs) as solid exchange medium and complex sample droplet to demonstrate the detailed operation processes of suspending, depositing, and separating (Fig. 1c and Supplementary Video 1). When the droplet propelled by light reaches the preloaded band of the MNPs in the PTM, the vortex generated during droplet motion suspends and disperses the MNPs homogeneously into the droplet. The MNPs deposit rapidly from the droplet with the aid of magnetic field. The supernate droplet is removed and separated from the MNPs fixed by the magnetic field.
In light of the photo/magnet-combined control strategy, we fabricated a PTM aiming at manipulation of solid/liquid mixed phases for protein analysis. The PTM was designed according to four criteria: (i) flexible; (ii) asymmetric deformation; (iii) wetting to aqueous solution; (iv) ultralow nonspecific adsorption. To satisfy the first criterion, the commercially available ethylene-vinyl acetate (EVA) copolymer microtube (with outer and inner diameters of 1000 µm and 800 µm, respectively) is chosen as the supporting layer for its good flexibility and comparable modulus. Our designed LLCP with azobenzene and biphenyl side chains in a 1:2 ratio (see Supplementary Figs. 1, 2 and 3 for synthetic routes) is coated on the inner wall of the EVA microtube to satisfy the second criterion. As shown in the cross section photographs of the PTM, the exposed surface of the PTM is displaced by 36.9 µm toward the 470 nm light (80 mW cm-2) driven by the deformation of the LLCP layer, which is attributed to reorientation of mesogens triggered by Weigert effect (Supplementary Figs. 4 and 5).34–37 Upon localized irradiation, the regional geometric change of the LLCP/EVA microtube from cylinder to conical structure generates Laplace pressure to propel the liquid slug.31 The last two criteria are satisfied by modifying the LLCP layer with bovine serum albumin (BSA), a commonly used blocking protein with excellent hydrophilicity in the field of biochemical analysis.37 Since reagents used in protein analysis are all aqueous solution, the BSA layer enhances the hydrophilicity to increase the driving force for propelling the aqueous droplets by Laplace pressure. Besides, BSA plays an important role as a blocking layer to decline nonspecific absorption rate, thus reducing the detection error during protein analysis.
The detailed fabrication processes of the PTM are described in Supplementary Fig. 6. The PTM exhibits ability to manipulate aqueous droplets of protein analysis reagents by light. As an example, a 5 µL droplet of avidin protein solution retreats from the irradiation site when the PTM is exposed to localized irradiation of 470 nm light (80 mW cm-2), and reverses the moving direction by altering the irradiation to the other side (Fig. 2a). Moreover, other kinds of protein analysis reagents, including phosphate buffer saline (PBS), MNP suspension, and fluorescence solution, are all manipulated by 470 nm light (80 mW cm-2) in the PTM, traveling at speeds ranging from 0.20 to 0.25 mm s-1 (Fig. 2b). The speeds of droplet motion increase gradually with the increase of light intensity, and the maximum speed of various protein analysis reagents reaches 0.4 mm s-1 (Supplementary Fig. 7).
Figure 2c shows the mechanism of photocontrolled droplet motion in the PTM. The diameter of the exposed wall increases owing to the photodeformation of the LLCP layer, while the unexposed part remains unchanged. Laplace pressure caused by this asymmetric shape change propels the wetting droplets with controllable speed and direction. It should be noted that the photocontrolled motion of aqueous droplets is attributed to the increase of driving force (Fd) and the decrease of resistance (Fr) after the BSA modification. Fd of the Laplace pressure correlates positively with the wettability of the droplets on the inner wall, and the Fr is related to the adhesion force (Fa) between the droplet and the inner wall. Contact angle (CA) measurements indicate that the CA of a 3 µL PBS droplet on the PTM inner wall decreases from 90.7° ± 2.5° to 37.5° ± 3.7° (Fig. 2d); thus, Fd increases with the enhanced hydrophilicity of the inner layer after the BSA modification. The Fa evaluation curve shows that the maximum Fa of the inner layer exerted on 3 µL PBS droplets decreases from 0.339 ± 0.031 mN to 0.259 ± 0.043 mN (Fig. 2e), suggesting the reduction of Fr. According to scanning electron microscope images (Supplementary Fig. 8), BSA on the inner wall forms dendritic crystals, whose microstructures reduce the contact area as well as Fa consequently between the droplets and the PTM.38 Therefore, aqueous droplets are propelled in the PTM by combining the asymmetric photodeformation of the LLCP layer and enhanced hydrophilicity of the BSA layer.
According to the photo/magnet-combined control strategy, a homemade prototype of PPA was constructed by the PTM, adapter, and mini-fluorimeter according to the strategy of photo/magnet-combined control (Supplementary Fig. 9). We demonstrated the extraction and detection of target avidin protein from a complex sample in the PPA with biotin-MNPs as solid exchange medium by fluorescence attenuation method (Fig. 3a). The preparation of the biotin-MNPs is described in Supplementary Figs. 10, 11 and 12.39 As Fig. 3b shows, a 5 µL complex sample is loaded to suspend the biotin-MNPs homogeneously, which capture the target avidin protein and then are separated from the supernate droplet with the aid of photo/magnet-combined control (i. extraction step). In sequence, a 5 µL droplet of biotin-4-fluorescence detection solution, a reported reagent for avidin detection,40, 41 is loaded to suspend the biotin-MNPs again. The fluorescence molecules are specifically bonded to the target avidin protein captured on the biotin-MNPs. After depositing and separating processes, the supernate droplet is exported to detect the attenuation degree of fluorescence signal (ii. detection step). To detect the fluorescence signal of the supernate droplet, the PTM is connected to an adapter through an L-shaped quartz tube, where the supernate will be propelled to the detection window from the PTM (Fig. 3c). The adapter is inserted into a mini-fluorimeter for measuring the fluorescence signal (λEx is 460 nm and λEm is 525 nm). Then, a smart phone is used to convert the fluorescence signal in to the concentration information of the target avidin protein in the samples.
The photo/magnet-combined control strategy provides reliable and time-saving detection results of avidin concertation, the standard curve of which possesses a fitting constant (R2) up to 0.99998 (Supplementary Fig. 13). The reliable detection results originate from the BSA layer, whose nonspecific adsorption rate is as low as 1.6% (Supplementary Fig. 14).42 The capture efficiency of the avidin by our strategy is much higher than that induced by conventional microfluidics. As shown in Fig. 4a, 100% avidin is rapidly captured from the complex sample in only 12 min by using the vortex flow, while the time for the same capture rate in the laminar flow prolongs to 30 min.
The high capture rate in the PTM is ascribed to the vortex, which is simulated by finite element analysis method (Fig. 4b). As shown in the velocity field of the moving droplet, the flow velocity in the center of the droplet is much higher than that at the edge, because the edge flow is hindered by the PTM viscous resistance. Non-uninform distribution of the flow velocities leads to that the fluid in the center tends to flow to the edge of the droplet, thus forming vortex flow trajectories.43 The resultant vortex suspended the biotin-MNPs in homogeneously, and increased the collision probability between the biotin-MNPs and the avidin, accelerating the capture procedure.
The droplet in the conventional microfluidics driven by syringe-pumps exhibited laminar flow, where the solid exchange mediums were difficult to be dispersed uniformly, owing to small channel dimensions and uniform flow rates.44 In order to generate the vortex, sophisticated channels, including herringbone, zigzag, and serpentine, were designed to increase channel dimensions and disturb flow rates in the conventional microfluidics.45–49 However, geometric change of the PTM from column to cone spontaneously gives rise to the vortex, resulting from the orientation change of the mesogens upon 470 nm light. All the operations of liquid/solid mixed phases, including suspending, depositing, and separating, are facilely manipulated in the milligram-level, 3 cm long PTM by a commercial laser pointer and a magnet.
The extraction and detection of the target proteins are completed in the PPA in the absence of external mechanical units of the conventional microfluidics.50–54 We comprehensively investigated the instrument parameters of our homemade PPA and a commercial microplate reader with enzyme-linked immunosorbent assay (ELISA) kits (see Supporting Information for detailed steps of protein analysis), which were summarized in Table 1. The egg white and cream were chosen as complex samples to analyze the content of the avidin by using both the PPA and microplate reader, respectively. Compared to the commercial microplate reader, the PPA features comparable detection accuracy and further shorten the analysis time from 90 to 20 min. The avidin antibodies fixed on the bottom of the ELISA kit captured the avidin by free diffusion (slow), while the biotin-MNPs were suspended by the vortex in the PPA to accelerate the reaction with the avidin (fast). The sample consumption of the PPA is only 5 µL, which is 10 times smaller than that of the microplate reader (50 µL), since the PPA is a droplet-based microfluidic system, where all the processes of protein analysis are operated inside microscale droplets. Moreover, the weight of the PPA is 400 g, accounting for approximately 1/40 of that of the microplate reader.
Table 1
Comparison of the parameters between the commercial microplate reader and the PPA.
|
Microplate reader
|
PPA
|
Solid exchange mediums
|
Avidin antibody
|
Biotin-MNPs
|
Detection result of egg white (µg mL− 1)
|
47 ± 3
|
51 ± 4
|
Detection result of cream (µg mL− 1)
|
33 ± 2
|
29 ± 3
|
Sample consumption (µL)
|
50
|
5
|
Total detecting time (min)
|
90
|
20
|
Equipment weight (kg)
|
15
|
0.4
|
Equipment volume (mm3)
|
500 × 300 × 290
|
194 × 155 × 72.5
|
In addition to protein extraction, the homemade PPA has the ability to enrich protein samples with low concentrations, which enables the dilute samples beneath the detection limit to be analyzed. For example, C-reactive protein (CRP), a biomarker of inflammation and cardiovascular disease, has a very low concentration of only 0.8-8 µg mL-1 in blood.50 We demonstrated the decline of the CRP detection limit after enrichment in the PPA by using 1°Ab-MNPs as solid exchange medium and 2°Ab-FITC as detection solution. The 1°Ab-MNPs captured the CRP from different amounts of 5 µL dilute samples loaded into the PPA sequentially. In this scenario, the CRP began to gradually accumulate on the 1°Ab-MNPs after enrichment, i.e. to repeat the extraction step for several times. These captured CRP were further specifically bonded to the 2°Ab-FITC in the 5 µL detection solution, whose supernate droplet was exported to detect the attenuation degree of fluorescence signal (Supplementary Fig. 15). As a result, the detection limit decreases from 9 to 1 µg mL-1, as we increase the extraction times to concentrate the dilute CRP samples from 45 to 5 µL (Fig. 5a). The standard curve with R2 up to 0.99983 after enrichment for 9 times spans 1–9 µg mL-1, covering the concentration range of the CRP in clinical samples (Fig. 5b). Therefore, our homemade PPA provides a versatile platform for fast and accurate protein analysis of various trace complex samples in different occasions.