2.1 Materials
DMSA Coated Fe3O4 Nanoparticles with a diameter of 20 nm were offered by Nanjing XFNANO Materials Tech Co., Ltd. (Jiangsu, China). Anti-cTnI McAb (coating) (2 mg mL-1) was purchased from Shanghai Linc-Bio Science Co., Ltd. (Shanghai, China). The cTnI antigen standard, concentrated biotin-labeled cTnI antibody (100×), concentrated HRP-labeled streptavidin (100×), antigen standard diluent, biotin-labeled cTnI antibody diluent, HRP-labeled streptavidin diluent, concentrated wash solution (25×), tris buffered saline (PH7.4, 20×), and Pluronic F-127 were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). BeyoECL Moon (Extreme Μltra Sensitive ECL Chemiluminescence Kit), Bovine serum albumin (BSA), and N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) were obtained from the Beyotime Institute of Biotechnology Co., Ltd. (Shanghai, China). N-hydroxysuccinimide (NHS) and phosphate buffer solution (PBS, PH 7.4) were provided by Sigma-Aldrich Co., Ltd. (Shanghai, China). MES (PH6.0, 0.1M) buffer was purchased from Vorradex (Fujian, China). Dimethyl silicone oil (5.0 CST) was purchased from Chiya Industrial Zone (Guangdong, China). Polydimethylsiloxane (PDMS) and curing agents were offered by Deji Trading Co., Ltd. (Shanghai, China). Indium tin oxide (ITO) glass chips were obtained from HuaNan XiangCheng Technology Co., Ltd. (Shenzhen, China). The FluoroPel PFC1601V was purchased from Cytonix (America). Photomultiplier tube (PMT), model H9306-04, was obtained from Hamamatsu Optoelectronics Co., Ltd. (Japan). The vortex oscillator was purchased from Changzhou Enpei Instrument Manufacturing Co., Ltd. (Jiangsu, China). The capture card (USB-6008) was obtained from National Instruments Co., Ltd. (America).
2.2 Methods
2.2.1 Design of DMF chips
The DMF chips were made up of two boards: the substrate and the top board. The substrate was the DMF driver chip, which was designed as an ITO glass with an electrode array pattern. Each electrode was a square with a side length of 1.9 mm. Each ITO electrode was led off by a corresponding ITO lead. ITO patterns were created in Auto CAD and then processed by HuaNan XiangCheng Technology Co., Ltd. The DMF driver chip for immunoassay was divided into five distinct functional domains: 1. Injection area for samples. 2. Area for washing solution injection. 3. Incubation and detection zone. 4. The injection site for chemiluminescent reagents. 5. Storage area for waste solution. Fig 1a displayed the five functional areas of the DMF substrate chip.
To facilitate the access of the DMF driver chip to the control circuit, a connecting finger array of ITO (pitch of 2.54 mm) was created, which was connected to the ITO electrodes. The DMF driver chip was then linked to the high voltage output circuit through two connecting finger slots. The DMF driver chip was connected to the circuit in the manner shown in Fig 1b.
PDMS and curing agents were mixed thoroughly in a ratio of 10:1 and then vacuumed for 20 minutes to eliminate air bubbles. A layer of PDMS was spin-coated onto the DMF driver chip at 4000 rpm for 5 minutes (10 μm) (Koschwanez et al., 2009). Then, the DMF driver chip was placed on the heating table and baked at 110 degrees for 10 minutes to cure the PDMS and obtain the PDMS film, which was treated as the dielectric layer. Following that, a layer of PFC1601V was spin-coated onto PDMS at 3800 rpm for 30 seconds, which functioned as the hydrophobic layer.
A block of ITO glass was utilized as the top plate for the DMF chips, and a layer of PFC1601V was spin-coated as the hydrophobic layer. Two thicknesses of copper foil (120 μm) separated the top and bottom plates. Before the use of the DMF chips, they were filled with silicone oil (5.0 CST) to serve as the medium for the movement of the reagents. The top plate was grounded with a wire when the DMF chips worked.
2.2.2 Design of the heating module
The heating pad (10 mm × 10 mm, 5 V, 1.36 w) was manufactured of polyimide heating film and was attached to the backside of the sample reaction area of the DMF driver chip with 3M double-sided tapes. The temperature was collected by the PT100 temperature sensor and was controlled by the REX-C700 temperature controller.
2.2.3 Design and operation of DMF detection system.
DMF detection systems were made up of two parts: the DMF drive system and the chemiluminescence detection system.
The master chip was the stm32f103vet6, which was programmed using MDK to control the complete drive circuit. The high voltage module was the KDHM-H-12S500P-20, which could produce a high voltage ranging from 0 to 500 V. The voltage output was regulated by the MCP41010, an integrated digital potentiometer. The DC-to-AC circuit consisted of an inverter (74LS14) and high-speed optocouplers, and the AC frequency was controlled by the PWM waveform of the primary control chip. The high-voltage output controller was the HV7224, a low-voltage serial to high-voltage parallel converter with push-pull outputs that could be configured for the activation and deactivation of 40 high-voltage circuits. The physical diagram of the DMF drive system was shown in Fig 2.
The chemiluminescence detection system consisted of an objective lens, PMT, USB-6008 acquisition card, and Labview software. The focus of the objective lens was aimed at the sample reaction area of the DMF driver chip, to capture more exact values of the luminous intensity in the sample reaction area. The chemiluminescence of the sample area was converted to parallel light by passing through the objective lens and was received by the PMT. The chemiluminescence signals were transformed into electrical signals by the USB-6008 acquisition card and displayed on the Labview software interface, and the light intensity values were recorded by Labview. Fig 3 illustrated the schematic diagram of the chemiluminescence assay system.
2.2.4 Preparation of cTnI antibody-coated magnetic beads
MES buffer was used to prepare EDC (50 mg mL-1) and NHS (50 mg mL-1) solution. The 20 μl magnetic beads (4 mg mL-1) were taken in a centrifuge tube and shaken for 15 seconds by the vortex oscillator to fully suspend the beads in the aqueous solution. After washing the beads 3 times with MES buffer, the supernatant was removed using a pipette, and then the beads were resuspended in 36 μl of MES buffer. Then, the suspension of magnetic beads was mixed with 12 µl EDC and 12 µl NHS for 20 minutes to activate the beads. A magnet was placed at the bottom of the centrifuge tube and after the magnetic beads were aggregated, the upper layer of unreacted EDC and NHS solution was sucked off with the pipette. The magnetic beads were washed once with 50 μl PBS and subsequently dispersed in 36ul of PBS buffer. Then, 4 μl anti-cTnI McAb (coating) (2 mg mL-1) was added to the solution of magnetic beads and reacted at room temperature for 2 hours to combine the magnetic beads. After that, 10 μl of 10% BSA in PBS was inserted for 30 minutes to block any possible residual sites on the magnetic beads. The supernatant was removed and the obtained beads were resuspended in 80 μl of 1% BSA in PBS to obtain anti-cTnI antibody-coated magnetic beads (1 mg mL-1) and stored at 4° C for further usage.
2.2.5 Optimization of DMF in immunoassay
The cTnI immunoassay reagents mainly include cTnI antibody-coated magnetic beads, cTnI antigen, biotin-labeled cTnI antibody, and HRP-labeled streptavidin. Protein-containing droplets and biological reagents are hydrophilic to DMF chips, which not only cause protein loss but also increase the difficulty of droplet manipulation.
The most efficient way of reducing protein adsorption is to add a certain amount of surfactant to the bioreagents. The use of Pluronic F-127 has been reported to improve electrowetting and decrease protein adsorption (Au et al., 2011; Sun et al., 2022). When compared to DMF without surfactant, the addition of Pluronic F-127 can boost the concentration of mobile reagents by up to 1000 times (Luk et al., 2008). All the reagents needed in the immunoassay experiment were added with 0.1 wt% Pluronic F-127 dissolved in TBS.
2.2.6 Design of DMF-based immunoassay
Reagents preparation. Sample dilutions for cTnI antigen standards were prepared at 25, 12.5, 6.25, 3.13, 1.56, 0.78, and 0.39 ng mL-1 concentrations of cTnI to be detected. The biotin-labeled cTnI antibody and HRP-labeled streptavidin were diluted by 100 times, respectively. The concentrated wash solution was diluted by 25 times with deionized water. Equal volumes of chemiluminescent reagents A and B were mixed for further use.
The immunoassay process was divided into 4 steps.
1. Injection and incubation of reagents. First, the cTnI antibody-coated magnetic bead droplet was driven by DMF from the reagent injection area (area 1) to the incubation area (area 3) of the DMF driver chip. Then, the cTnI antigen, biotin-labeled cTnI antibody, and HRP-labeled streptavidin were sequentially moved from the reagent injection area to the incubation area and mixed. As shown in Fig 4a, the droplet in the DMF chips was moved towards the incubation area. After each addition and incubation of the reagents for 10 minutes (2 µl of each reagent), the unbound parts were removed by the DMF driver chip.
2. Extraction of magnetic beads. A magnet was placed under the DMF driver chip manually, and after the magnetic beads were gathered, the droplets were moved to the waste storage area. The extraction of magnetic beads from the sample droplet was represented in Fig 4b.
3. Washing. Five drops of washing solution, each of 2 µl, were injected sequentially from the washing solution storage area, and the magnetic beads were washed five times.
4. Detection. 2 μl of chemiluminescent reagents were injected from the chemiluminescent reagents feed zone. The washed magnetic beads were combined with the chemiluminescent reagents and moved to the detection area for analysis. The formation of the magnetic beads-based enzyme-labeled complexes was shown in Fig 5.
The immunoassay process was performed in a dark box. Chemiluminescence intensity data were collected on the computer by Labview. The whole detection procedure was completed within 40 minutes.