阅读说明：本技术 一种大肠癌免疫传感器的制备及其检测方法 (Preparation and detection method of colorectal cancer immunosensor ) 是由 刘洋 黎玉凤 李旺 于 2019-09-09 设计创作，主要内容包括：本发明公开了本发明提供了一种大肠癌免疫传感器的制备及其检测方法,采用如下步骤制备：采用绝缘硅片作为底层；制备半导体层和导电层；刻蚀呈三角分布的槽孔,三角分布的槽孔分别作为免疫传感器的源电极、漏电极和栅电极；形成了半导体硅与导电层之间的欧姆接触；用采用电感耦合等离子体刻蚀技术制作出多个微流控通道、液体出口通道和入口通道,清除残留物；进行非特异性活性处理,保存于3-4℃温度下备用,得到免疫传感器。传感器在检测应用上,灵敏度高、准确度高,能实时反应且免于标记。(The invention discloses a preparation method and a detection method of a colorectal cancer immunosensor, which are prepared by the following steps: adopting an insulating silicon wafer as a bottom layer; preparing a semiconductor layer and a conductive layer; etching triangular distributed slotted holes which are respectively used as a source electrode, a drain electrode and a gate electrode of the immunosensor; forming an ohmic contact between the semiconductor silicon and the conductive layer; manufacturing a plurality of microfluidic channels, liquid outlet channels and liquid inlet channels by using an inductively coupled plasma etching technology, and removing residues; performing nonspecific activity treatment, and storing at 3-4 deg.C to obtain immunosensor. The sensor has high sensitivity and high accuracy in detection application, can react in real time and is free from marking.)

1. A preparation method of a colorectal cancer immunosensor is characterized by comprising the following steps:

(1) adopting an insulating silicon wafer as a bottom layer;

(2) depositing a silicon dioxide (SiO 2) sheet with the thickness of 15nm on the surface of an insulating silicon wafer to form a semiconductor layer by a plasma enhanced chemical vapor deposition method, and then using titanium dioxide as a conductive layer by a chemical vapor deposition process and passivating the conductive layer;

(3) etching triangular-distributed slots on the surface of the insulating layer by using a Nikon stepping photoetching instrument, wherein the triangular-distributed slots are respectively used as a source electrode, a drain electrode and a gate electrode of the immunosensor, and a conducting layer between the source electrode and the drain electrode is disconnected by arranging a spacing layer between the source electrode and the drain electrode so as to be insulated;

(5) forming a source electrode, a drain electrode and a gate electrode in the hole groove, preparing the source electrode, the drain electrode and the gate electrode by a metal deposition method, and then treating the source electrode, the drain electrode and the gate electrode by adopting an acidic solution;

(6) carrying out rapid thermal annealing treatment on the semi-finished product prepared in the step 1-5 in an environment of 410-450 ℃ to form ohmic contact between the semiconductor silicon and the conductive layer;

(7) manufacturing a plurality of microfluidic channels, liquid outlet channels and liquid inlet channels by adopting an inductively coupled plasma etching technology, and removing residues;

(8) and (3) putting the sensor prepared by 1-7 into an absolute ethanol solution containing 4% of 3-Aminopropyltriethoxysilane (APTES) for 20 minutes, rinsing the modified electrode by using a Phosphate Buffer Solution (PBST) containing 0.4%, dripping 20 mu L of 6% bovine serum albumin, sealing the nonspecific active site on the electrode, and storing at the temperature of 3-4 ℃ for later use to obtain the immunosensor.

2. The method of claim 1, wherein the silicon wafer is square or circular with thin edges and thick middle, and the insulating sheet has a thickness of 40-60 nm.

3. The method of claim 1, wherein the conductive layer has a thickness of 6-15 nm, and the conductive layer is connected to the probe of the semiconductor detection device via a nanowire.

4. The method of claim 1, wherein the spacer is etched to form a recess between the source and the drain.

5. The method of claim 1, wherein the microfluidic channel is disposed between the semiconductor layer and the conductive layer, the liquid outlet channel and the liquid inlet channel are connected to the conductive layer and are connected to two ends of the microfluidic channel, the microfluidic channels are arranged in parallel, and the diameter of the microfluidic channel is 3-6 μm.

6. A method for detecting a colorectal cancer immunosensor, comprising the steps of:

(1) preparing a matched instrument, and connecting an instrument probe with the sensor of claims 1-5;

(2) and obtaining the concentration of the detected object in the sample according to the standard curve corresponding to the measured reduction peak current value.

Technical Field

The invention relates to the technical field of medical detection, in particular to preparation of a colorectal cancer immunosensor and a detection method thereof.

Background

Since death due to colon cancer is increasing, and the 5-year survival rate is very high when colon cancer is found at an early stage and appropriately treated as compared with other cancers, mass screening (mass screening) of colon cancer is one of the most effective methods.

For confirmation of colon cancer, an endoscopic examination is generally performed in which the colon is directly visually confirmed, and if necessary, a biopsy of the affected part is performed. However, these methods are invasive and require a high degree of expertise and are therefore not suitable for such primary screening in population screening.

As a method for non-invasive detection of colon cancer, there is a method using a component contained in feces as an index. Since feces contain cells exfoliated from cancer tissues, the composition of feces is thought to reflect digestive tract disorders. Therefore, colon cancer is detected by using a gene which is hardly expressed in normal tissues but highly expressed in cancer tissues as a biomarker, using the amount of mRNA of the gene in feces, carcinoembryonic antigen (CEA), CA19-9, and the like as indicators, and using the amount of the gene in feces as an indicator. However, there are problems in that: the sensitivity using these biomarkers is substantially the same or lower than that of the fecal occult blood method. In particular, any of the above biomarkers has a lower sensitivity for detection of early stage cancer and progressive adenoma than does occult blood. Therefore, development of a method free from labeling and high in sensitivity is strongly desired.

Disclosure of Invention

The invention aims to provide a preparation method of a colorectal cancer immunosensor, which solves the problems in the prior art.

In order to achieve the purpose, the invention provides a preparation method of a colorectal cancer immunosensor, which is characterized by comprising the following steps:

(1) adopting an insulating silicon wafer as a bottom layer;

(2) depositing a silicon dioxide (SiO 2) sheet with the thickness of 15nm on the surface of an insulating silicon wafer to form a semiconductor layer by a plasma enhanced chemical vapor deposition method, and then using titanium dioxide as a conductive layer by a chemical vapor deposition process and passivating the conductive layer;

(3) etching triangular-distributed slots on the surface of the insulating layer by using a Nikon stepping photoetching instrument, wherein the triangular-distributed slots are respectively used as a source electrode, a drain electrode and a gate electrode of the immunosensor, and a conducting layer between the source electrode and the drain electrode is disconnected by arranging a spacing layer between the source electrode and the drain electrode so as to be insulated;

(5) forming a source electrode, a drain electrode and a gate electrode in the hole groove, preparing the source electrode, the drain electrode and the gate electrode by a metal deposition method, and then treating the source electrode, the drain electrode and the gate electrode by adopting an acidic solution;

(6) carrying out rapid thermal annealing treatment on the semi-finished product prepared in the step 1-5 in an environment of 410-450 ℃ to form ohmic contact between the semiconductor silicon and the conductive layer;

(7) manufacturing a plurality of microfluidic channels, liquid outlet channels and liquid inlet channels by adopting an inductively coupled plasma etching technology, and removing residues;

(8) and (3) putting the sensor prepared by 1-7 into an absolute ethanol solution containing 4% of 3-Aminopropyltriethoxysilane (APTES) for 20 minutes, rinsing the modified electrode by using a Phosphate Buffer Solution (PBST) containing 0.4%, dripping 20 mu L of 6% bovine serum albumin, sealing the nonspecific active site on the electrode, and storing at the temperature of 3-4 ℃ for later use to obtain the immunosensor.

Preferably, the silicon-on-insulator sheet is square or round with thin edges and thick middle, and the thickness of the insulating sheet ranges from 40nm to 60 nm.

Preferably, the thickness of the conductive layer is between 6 and 15 nanometers, and the conductive layer is connected with a probe of the semiconductor detection device through a connecting nanowire.

Preferably, the spacer layer may be formed by recessing between the source and the drain by etching.

Preferably, the microfluidic channel is arranged between the semiconductor layer and the conductive layer, the liquid outlet channel and the liquid inlet channel are communicated with the conductive layer and are respectively communicated with two ends of the microfluidic channel, the microfluidic channels are distributed in parallel, and the diameter range of the microfluidic channels is 3-6 μm.

Another object of the present invention is to provide a method for detecting a colorectal cancer immunosensor, including the steps of:

(1) preparing a matched instrument, and connecting an instrument probe and a sensor;

(2) and obtaining the concentration of the detected object in the sample according to the standard curve corresponding to the measured reduction peak current value.

Compared with the prior art, the invention has the following beneficial effects:

1. the method is adopted to form the test object and the sensor in a cross grid structure, so that the cross interconnection of the nanowires is realized. In the triangular bridging grid structure, when biomolecules pass through between two branches, light waves and a disturbed electric field are mutually shielded, so that the light intensity and the current detected by the sensor are changed, and the detection of liquid samples (such as biomolecules, cells and the like) is realized.

2. The micro-fluidic channels are uniformly arranged, so that the micro-fluidic channels have large specific surface area and can effectively collect and enhance signals.

3. The 3-aminopropyltriethoxysilane and other substances react in a covalent bonding mode to modify and resist specificity, rather than the traditional electrostatic adsorption effect, so that the nonspecific adsorption resistance is improved, qualitative and quantitative detection can be realized by promoting the positive correlation between the sensor and a detected substance, and the sensitivity is high.

4. The sensor of the invention has high sensitivity and high accuracy in detection application, can react in real time and is free from marking.

Detailed Description

The following detailed description of the present invention will be given with reference to examples, but it should be understood that the scope of the present invention is not limited to the specific embodiments.

The colorectal cancer immunosensor disclosed by the invention is prepared by the following steps:

(1) an insulating silicon wafer is used as a bottom layer, the insulating silicon wafer is preferably square or round with thin edges and thick middle, the thickness range is 40-60 nm, the edge is preferably 40nm, and the middle is 60nm thickest.

(2) Depositing a silicon dioxide (SiO 2) thin sheet with the thickness of 15nm on the surface of an insulating silicon wafer by a plasma enhanced chemical vapor deposition method to form a semiconductor layer, then taking titanium dioxide as a conductive layer by a chemical vapor deposition process, and passivating the conductive layer, wherein the thickness of the conductive layer is between 6 and 15 nanometers, preferably 12 nanometers, and the conductive layer is used for connecting the nanowire with a probe of a semiconductor detection device.

(3) Etching triangular slotted holes on the surface of the insulating layer by using a Nikon stepping photoetching instrument, preferably forming a close-packed array so as to place nano particles; the slots distributed in a triangular shape are respectively used as a source electrode, a drain electrode and a gate electrode of the immunosensor, wherein a conducting layer between the source electrode and the drain electrode is disconnected by arranging a spacing layer between the slots of the source electrode and the drain electrode so as to be insulated, and the spacing layer can be realized by forming a groove between the source electrode and the drain electrode by adopting an etching method or in other modes.

(5) And forming a source electrode, a drain electrode and a gate electrode in the hole groove by a metal deposition method, and then treating by adopting an acidic solution.

(6) And carrying out rapid thermal annealing treatment on the semi-finished product prepared in the steps 1-5 in an environment of 410-450 ℃ to form ohmic contact between the semiconductor silicon and the conductive layer.

(7) Manufacturing a plurality of microfluidic channels, liquid outlet channels and liquid inlet channels by adopting an inductively coupled plasma etching technology, and removing residues; the microfluidic channels are arranged between the semiconductor layer and the conductive layer, the liquid outlet channels and the liquid inlet channels are communicated with the conductive layer and are respectively communicated with two ends of the microfluidic channels, the microfluidic channels are distributed in a parallel mode, and the diameter range of the microfluidic channels is 3-6 microns, preferably 3 microns or 6 microns.

(8) And (3) putting the sensor prepared by 1-7 into an absolute ethanol solution containing 4% of 3-Aminopropyltriethoxysilane (APTES) for 20 minutes, rinsing the modified electrode by using a Phosphate Buffer Solution (PBST) containing 0.4%, dripping 20 mu L of 6% bovine serum albumin, sealing the nonspecific active site on the electrode, and storing at the temperature of 3-4 ℃ for later use to obtain the immunosensor.

The method is adopted to form the test object and the sensor in a cross grid structure, so that the cross interconnection of the nanowires is realized. In the triangular bridging grid structure, when biomolecules pass through between two branches, light waves and a disturbed electric field are mutually shielded, so that the light intensity and the current detected by the sensor are changed, and the detection of liquid samples (such as biomolecules, cells and the like) is realized. In addition, the micro-fluidic channels are uniformly arranged, so that the micro-fluidic channels have large specific surface area and can effectively collect and enhance signals. In addition, 3-aminopropyltriethoxysilane and other substances react in a covalent bonding mode to modify and resist specificity, rather than the traditional electrostatic adsorption effect, so that the capacity of resisting nonspecific adsorption is improved, qualitative and quantitative detection can be realized by promoting the positive correlation between the sensor and a detected substance, and the sensitivity is high.

The detection method of the colorectal cancer immunosensor comprises the following steps:

(1) and (4) preparing a matched instrument, and connecting the probe of the instrument with the sensor.

The prepared instrument at least comprises: the device comprises a Nikon NSR 1755i B stepping photoetching instrument, an Agilent B1500A semiconductor parameter measuring instrument, a multi-path high-precision peristaltic pump, a KQ5200 type ultrasonic cleaner, a 2XZ-1 type rotary vane vacuum pump, a digital display temperature control electric heating plate, a constant temperature oscillator, an electric heating constant temperature blowing drying box and a laboratory water purification system, wherein the connection relation of the devices is realized by adopting the prior technical scheme and a general technical scheme, and when the device is connected with a sensor, insulation, waterproof and anti-pollution treatment is paid attention to, and the prepared sensor can be used after being sealed and connected.

(2) And obtaining the concentration of the detected object in the sample according to the standard curve corresponding to the measured reduction peak current value.

For example, detection of RNA solutions:

the sealed sensor is connected with a multi-path high-precision peristaltic pump and a semiconductor parameter measuring instrument, and the peristaltic pump continuously pumps the RNA antigen solution into a micro-channel of the sensor from a test tube at the speed of 18 mu L/min. Each test result is independently tested by three groups of probes, and the test process is carried out in a room temperature environment (25 +/-2 ℃).

In the detection experiment, when RNA antigen solutions with different concentrations flow into a sensing window of Si-NR through a microfluidic pore channel, the voltage applied to a grid electrode is-5V in the whole detection process, so that an electric field can be uniformly distributed. In the detection of the concentration of RNA, RNA solutions having concentrations of 10pg/ml, 100pg/ml, 1ng/ml, 10ng/ml and 100ng/ml, respectively, were circulated in the microfluidic channel for 100 seconds, and a real-time current-time curve of NR was obtained. The experimental result clearly shows that the Ids saturation current increases along with the increase of the concentration of the RNA antigen solution, and the sensor has high sensitivity and high accuracy, can react in real time and is free from marking in the detection application.

The detection of a gene solution such as RNA, BRAF, APC is also carried out by the method described above.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.