CN106596642A - Hydrophobic modification based blood coagulation sensor, preparation method and application thereof - Google Patents

Abstract

This case involves a coagulation detection sensor based on hydrophobic modification, its preparation method and application. The coagulation detection sensor includes a piezoelectric film sensor, and a hydrophobic layer is modified on the surface of the piezoelectric film sensor, and the hydrophobic layer is polyxylylene. In this case, Parylene was vacuum-evaporated onto the surface of the piezoelectric film sensor to improve the hydrophobicity of the surface and achieve the effect of adsorbing the hydrophobic domain of fibrin; it reduced the impact on the piezoelectric sensor and enhanced the binding of the sensor surface to the coagulation end product fibrin ability to achieve the effect of improving the frequency stability of the sensor output signal; in addition, this case has the characteristics of batch modification, which helps to eliminate the difference between batches of chip modification.

Description

Coagulation detection sensor based on hydrophobic modification, its preparation method and application

technical field

The invention relates to a coagulation detection sensor, in particular to a hydrophobic modification-based coagulation detection sensor, its preparation method and application.

Background technique

At present, optical detection methods have been widely used in blood coagulation detection. The most commonly used detection indicators in the test are prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen (FIB), thrombin time (TT). ), endogenous coagulation factors, exogenous coagulation factors, high-molecular-weight heparin, low-molecular-weight heparin, protein C, protein S, etc. There are many detection items, but optical detection has strict requirements on the samples to be tested, and the detection time is long, so there are certain limitations. The optical principle is mainly scattering turbidimetry: the detection end point is determined according to the change of scattered light of the sample to be tested during the solidification process. In this method, the monochromatic light source of the detection channel and the light detector are at a right angle of 90°. When the coagulation activator is added to the sample, the scattered light intensity of the sample gradually increases with the formation of the fibrin clot in the sample. When the sample is completely solidified, the intensity of the scattered light does not change. Usually, the starting point of solidification is regarded as 0%, the end point of solidification is regarded as 100%, and 50% is regarded as the solidification time. The optical detector receives this optical change, converts it into an electrical signal, and then transmits it to the monitor for processing after amplification to draw the coagulation curve. Therefore, when detecting complex samples with poor light transmission such as whole blood, optical The detection method cannot obtain correct detection results, and it is difficult to realize miniaturized and portable equipment by building an optical path based on optical principles.

Piezoelectric film sensor (QCM) is a high-sensitivity mass sensor. Its detection principle is that the blood to be measured changes from fibrinogen to fibrin and combines with the surface of the film sensor to produce viscoelastic changes that cause changes in resonance frequency. The response to nanogram-level mass change has the characteristics of high sensitivity and fast response speed. Compared with the optical coagulation instrument, the piezoelectric film sensor has fewer detection items, but it can realize multi-index detection through multiple channels, and the detection time is short. In addition, the piezoelectric film sensor has the characteristics of small size, which is easy to realize the development and production application of miniaturized equipment.

When the piezoelectric film sensor is directly used for the detection of complex samples such as serum or whole blood, it also faces the problem of complicated components in the sample system, and a large number of non-target proteins and soluble components will cause great background interference. Conventional solutions deposit affinity proteins such as protein A, streptavidin, and biotin on the sensor surface to improve the binding efficiency of specific antibodies or biomolecules. However, the method of antigen-antibody combination requires too long reaction time, which cannot meet the requirements of rapid coagulation detection (within 3 minutes). The normal value of aPTT is 28s-33s (varies according to different reagents), and the normal value of PT is 12s-16s. Therefore, it is necessary to develop a new blood coagulation detection sensor in this case to improve the adsorption specificity and shorten the detection time.

Contents of the invention

In view of the technical problems existing in the prior art, this case provides a coagulation detection sensor based on hydrophobic modification, its preparation method and application. By applying the material of parylene (Parylene) to the detection of coagulation time, the piezoelectric film is strengthened The surface modification method of sensor performance (signal-to-noise ratio, specificity, repeatability) to solve the problem of unstable detection frequency and long detection time of the chip caused by non-specific adsorption interference in the detection process of blood coagulation in the piezoelectric film sensor in the prior art .

In order to achieve the above purpose, this case is realized through the following technical solutions:

A blood coagulation detection sensor includes a piezoelectric thin film sensor, and a hydrophobic layer is modified on the surface of the piezoelectric thin film sensor, and the hydrophobic layer is polyxylene.

Preferably, in the coagulation detection sensor, the thickness of the hydrophobic layer is 100-1500 nm.

A method for preparing a blood coagulation detection sensor, which uses vacuum evaporation to plate a hydrophobic layer on the surface of a piezoelectric film sensor, and the hydrophobic layer is polyxylene.

Preferably, in the preparation method of the blood coagulation detection sensor, the thickness of the hydrophobic layer is 100-1500 nm.

An application of the coagulation detection sensor as described above in blood coagulation detection of serum samples.

An application of the coagulation detection sensor as described above in coagulation detection of whole blood samples.

The beneficial effects of the present invention are:

(1) In order to solve the problem of adsorbing fibrin produced by blood coagulation to the surface of the piezoelectric film sensor, Parylene was vacuum evaporated on the surface of the piezoelectric film sensor for the first time to improve the hydrophobicity of the surface and achieve the effect of adsorbing the hydrophobic domain of fibrin .

(2) Parylene, a nano-scale thickness material with good hydrophobicity, reduces the impact on the piezoelectric sensor, enhances the binding ability of the sensor surface to the coagulation end product fibrin, and realizes the effect of improving the frequency stability of the sensor output signal.

(3) Parylene itself is a strong acid and alkali resistant material, which has excellent ability to protect chips.

(4) The Parylene material can be modified in batches, which helps to eliminate the differences between batches of chip modification.

Description of drawings

Figure 1 is a comparison chart of the detection results of the activated partial thromboplastin time (aPTT) detected by the sensor chip modified by Parylene in this case and the detection result of the sensor chip of unmodified Parylene.

Figure 2 is a comparison chart of the results of the same Parylene-modified sensor chip in this case to detect the blood samples of the same patient three times.

Figure 3 is a comparison chart of the detection results of activated partial thromboplastin time (aPTT) detected by the sensor chip modified by Parylene in this case and the detection results of Lambda950 ultraviolet spectrophotometer.

Figure 4 is a comparison chart of the detection results of activated partial thromboplastin time (aPTT) detected by the sensor chip modified by Parylene in this case and the detection results of Sysmex 2000i.

detailed description

The present invention will be further described in detail below in conjunction with the accompanying drawings, so that those skilled in the art can implement it with reference to the description.

In this case, the material with hydrophobic properties, parylene, is covered on the surface of the piezoelectric thin film sensor by vacuum evaporation technology to form a dense hydrophobic film structure to detect blood clotting time, including prothrombin time PT, activation part Thromboplastin time aPTT and so on.

The specific preparation process is as follows: the solid powder of Parylene (a class of substances) is evaporated at 150°C and 1 Torr, and is cracked at 690°C and 0.5 Torr to form Parylene gaseous molecules, which reach the piezoelectric film sensor chip through the dredging pipeline. Vacuum reaction chamber, as the temperature in the chamber drops, Parylene gaseous molecules form a dense Parylene hydrophobic film structure on the surface of the piezoelectric film sensor. The thin-film piezoelectric chip modified by Parylene is ready for blood clotting time detection. In addition, the modification technology is carried out in a vacuum tank, which can be operated in batches, and is easy to realize the development and manufacture of miniaturized sensing technology products.

After placing the chip in the detection system, add a total of 100 microliters of the blood sample and aPTT mixture with a pipette gun, and then add calcium ions (50 microliters) to start the coagulation reaction. The end product of coagulation, fibrin, is a hydrophobic structural protein , when the coagulation is over, the fibrin will be cross-linked into a network structure to expose its hydrophobic domain to the maximum extent. According to the hydrophobic interaction, the hydrophobic domain of the protein will be adsorbed with the hydrophobic surface formed by Parylene modification, and finally due to The change of viscoelasticity causes the quality change on the surface of the piezoelectric film sensor, which responds to the frequency change, and the sensing signal is transmitted to the display screen to output the result.

As shown in Figure 1, a negative experiment without the addition of plasma or whole blood (curve 1) does not produce a significant frequency drop due to the absence of fibrin adsorption to the sensor surface. The two aPTT test results (curve 2) of the unmodified piezoelectric film chip show obvious frequency jitter, which is caused by the fibrin not firmly bound to the surface and floating in the liquid phase. The coagulation time test results of the Parylene modified chip (curve 3) are in sharp contrast to curve 2, and the stability is significantly improved, which benefits from the improvement of the specificity of the sensor surface to fibrin after hydrophobic modification. In addition, due to the frequency jitter (see the end of curve 2) of the unmodified chip detection result (curve 2), it is difficult to have repeatability of the experiment.

As shown in Figure 2, the three curves of the coagulation time of the same sample detected by the modified chip show good repeatability. On the one hand, it is because the hydrophobic surface formed by Parylene improves the specificity of fibrin; on the other hand, it is because Parylene material has a strong Good biocompatibility, it can be tested again only after washing with water, and the difference between batches is small.

As shown in Figure 3, the optical standard inspection aPTT curve 1 and the Parylene-modified chip detection aPTT curve 2, the corresponding coagulation time point (position 3) is in good agreement, which proves that this scheme is consistent with the standard detection method (YY/T0659-2008) The results were consistent.

As shown in Figure 4, this is a comparison chart of the linear relationship between the aPTT results of the Parylene-modified chip and the clinical testing instrument Sysmex2000i aPTT. It shows that the Parylene-modified QCM chip is highly consistent with the test results of existing clinical equipment and can be used as Medical instruments and equipment are used for aPTT detection in clinical medicine.

Although the embodiment of the present invention has been disclosed as above, it is not limited to the use listed in the specification and implementation, it can be applied to various fields suitable for the present invention, and it can be easily understood by those skilled in the art Therefore, the invention is not limited to the specific details and examples shown and described herein without departing from the general concept defined by the claims and their equivalents.

Claims (6)

1. A blood coagulation detection sensor, characterized in that it includes a piezoelectric film sensor, the surface of the piezoelectric film sensor is decorated with a hydrophobic layer, and the hydrophobic layer is polyxylene. 2. The blood coagulation detection sensor according to claim 1, wherein the thickness of the hydrophobic layer is 100-1500 nm. 3. A method for preparing a blood coagulation detection sensor, characterized in that a hydrophobic layer is plated on the surface of the piezoelectric film sensor by vacuum evaporation, and the hydrophobic layer is polyxylene. 4 . The method for preparing a coagulation detection sensor according to claim 3 , wherein the thickness of the hydrophobic layer is 100-1500 nm. 5. An application of the blood coagulation detection sensor according to claim 1 or 2 in blood coagulation detection of serum samples. 6. An application of the coagulation detection sensor according to claim 1 or 2 in coagulation detection of whole blood samples.