Materials: protocatechuic acid, PDMS-glass chip, CalceiAm fluorescent dye for sodium citrate vein blood vacuum shearer tube, RSP01-CS two-way push-pull precision injection pump, IXT1 inverted fluorescence microscope, plasma cleaner, CD42b antibody, dimethyl sulfoxide, acetylsalicylic acid, tirofiban, adenosinediphosphate, collagen, thromboelastograph, Kaolin activator, coagulation factor detection kit,SysmexCA7000 automatic coagulation analyzer and its supporting reagents JAPAN,and FITC-CD62p.
Fabrication of microfluidic chips
The microfluidic chip for platelet adhesion and aggregation analysis consists of narrow microchannels and sample pools on both sides and outlets (Fig. 1). The working principle is that the blood samples are loaded into the sample pool, and the negative pressure generated by the outlet controls the blood sample flow through the microchannels at a set shear rate, and platelet adhesion and aggregation behavior are observed in the microchannels. Microfluidic chips were fabricated by dry film soft lithography14. The chip mask pattern was designed by Coreldraw 12.0 software and printed on transparent film using an inkjet printer. The two-layer photosensitive dry film (single layer thickness 35 µm) was laminated on a glass plate using a film-covering machine. The photosensitive dry film was irradiated by ultraviolet light through the mask for 50 s, and was developed with 1% sodium carbonate to form the chip mask. The prepolymer mixed at a 10:1 weight was poured onto the dye of the chip, and the bubbles were removed by vacuum evacuation. The prepolymer was cured for 3 h at 60℃. The solidified PDMS substrate was peeled from the photosensitive dry film anode, and the sample pool and outlet were formed by punching with a flat-end puncher (7 and 1.5 mm in diameter). The sample pool and outlet were irreversibly bonded with the clean glass carrier after being treated by an oxygen plasma cleaner (30 W, 1 min) to form a PDMS-glass microfluidic chip, A schematic diagram of the experimental device are shown in Fig. 1A,B.
Blood sample collection
Blood samples were collected from 20 healthy volunteers recruited from the Physical Examination center of Yongchuan Hospital affiliated to Chongqing Medical University from March to June 2019, and 10 volunteer patients with atherosclerosis recruited from the Cardiovascular Medicine department. Selection criteria: Healthy volunteers: no history of medication, operations, or alcoholism within one month, and hematocrit, platelet count, coagulation function (PT, APTT) and thromboelastogram (R, K, Angle, MA, CI) were within the normal reference range. Volunteer patients with atherosclerosis: the patient was not treated surgically at first admission. This study was approved by the Ethics Committee of Yongchuan Hospital affiliated with Chongqing Medical University (No. 2018035), and all subjects provided written informed consent. Venous blood samples were collected using a vacuum, and were anticoagulated with 1:9 (v/v) 3.2% sodium citrate and used as soon as possible. To test the inhibition of platelet adhesion and aggregation, tirofiban hydrochloride of different concentrations was prepared with 10 ml 0.9% sodium chloride solution added to 1 ml blood sample as the drug treatment group, and 10 ml 0.9% sodium chloride solution only was added as the control group. Finally, 1 mmol/L calcein AM fluorescent dyes were added to blood samples at a concentration of 1:500 (v/v). The samples were shaken gently and incubated at 37 °C in an incubator for 15 min. Calcein AM, as a living cell fluorescent dye, penetrates cell membranes and enters the cell. Calcein emits strong green fluorescence after being sheared by intracellular esterase. Therefore, Calcein AM was used to fluorescently label platelets in blood samples.
Detection of platelet aggregation
Microfluidic chips were treated with plasma cleaner (30 W) for 2 min to increase their hydrophilicity. The modified microfluidic chips were placed on the carrier of the inverted fluorescence microscope. The chip outlet was connected to the injection pump with a polytetrafluoroethylene tube (inner diameter 1.0, outer diameter 1.5 mm). The flow shear rate of the blood samples in the microchannel was controlled by the pullback mode. The flow rates were 10, 50, and 100 ul/min, respectively. The relationship between the fluid shear rate and injection pump flow rate in the microchannel was calculated according to Poiseuille's law, i.e., v=6Q/a2b. Among them, V (s-1) represents shear rate, Q (ul/s) represents flow rate, a (mm) represents depth of microchannel, and B (mm) represents width of microchannel. When blood began to flow into the microchannels, Streampix 5.0 software was used to control the camera to record fluorescence-labeled platelet adhesion and aggregation on the collagen surface at a frame rate of 1 frame/s (objective, *20), and 180 fluorescent images were recorded for 3 min. A schematic diagram and photos of the experimental device are shown in Fig. 1.
Detection of platelet activation markers
The AM fluorescent dye-labeled sodium citrate anticoagulated whole blood sample was treated with 2 μl dimethyl sulfoxide, ADP, ADP+PAC, Ristomycin, Ristomycin+PAC, and PAC, and was sequentially collected via a microfluidic chip. The specimen at the exit was fixed in paraformaldehyde solution. The machine was tested within 24 h to detect the expression of platelet CD62p.
Detection of platelet aggregation induced by gradient shear force
AM fluorescein-labeled sodium citrate anticoagulated whole blood samples were divided into three groups, and dimethyl sulfoxide, CD42b, and tirofiban hydrochloride were added, respectively. Then, the mixtures were incubated at 37°C in darkness for 10 min, and the blood flow rate was adjusted (100 μl/min). When the blood began to flow into the microchannel, Streampix 5.0 software was used to control the camera to record the images of fluorescence-labeled platelet adhesion and aggregation on the collagen surface at a frame rate of 1 frame/s (objective lens, ×20), and a total of 300 frames were recorded for a 5 min sequence of fluorescent images. The degree of Platelet aggregation was then measured in a microfluidic chip device.
Detection of platelet aggregation after inhibitor action
Prepare a certain concentration of inhibitor，and 1 ml sodium citrate anticoagulated whole blood samples were treated with aspirin, tirofiban hydrochloride, CD42b, and procatechin acid with AM fluorescent dye, respectively, and were incubated at 37°C for 10 min in darkness. Then, The degree of platelet aggregation was measured in a microfluidic chip device.
Detection of platelet aggregation induced by inducers
Prepare a certain concentration of Inducer and 1 ml sodium citrate anticoagulated whole blood samples were treated with collagen (4μg/ml), ADP (16M), ristomycin (0.4mg/ml) then incubated at 37°C for 10 min in darkness. Then, The degree of platelet aggregation was measured in a microfluidic chip device.
Detection of blood coagulation function after drug action
The protocatechuic acid solution（8 μmol/L） of 2 μl was added into 1 ml whole blood solution and incubated at 37°C for 10 min. The coagulation function was detected by thromboelastograph.And then add 1ml of whole blood into the kaolin activator tube and mix well, take 340μl of whole blood into the thromboelastometer for detection, record the detection time and graph.Secondly, the blood was centrifuged at 1782g for 5 minutes, and the prothrombin time (PT) and activated partial thromboplastin time (APTT) of the plasma were collected and recorded in detail.
The recorded data were input using Excel software, and SPSS 19.0 was used for data analysis. The count data were expressed as mean ± calibration difference ( x ± s). Analysis of variance using completely randomized design data was statistically significant at P < 0.05.