Preparation method of HEMT (High Electron Mobility Transistor) sensor based on engineered S-layer and biomolecule detection system

Abstract

The invention discloses an engineered S-layer and a preparation method thereof, an HEMT sensor based on in-vitro self-assembly of the engineered S-layer and a preparation method of the HEMT sensor and a biomolecule detection system, the engineered S-layer is formed by fusion expression of S-layer protein based on recombination or redesign and a biological sensitive element, and the biological sensitive element is protein or polypeptide; the HEMT sensor based on engineering S-layer in-vitro self-assembly comprises a field effect transistor body and a grid sensitive region, the field effect transistor body comprises a source electrode and a drain electrode which are arranged on the two sides respectively. The grid electrode sensitive region is arranged between the source electrode and the drain electrode and comprises a semiconductor material for forming a heterojunction structure and a two-dimensional protein nano array containing a biological sensitive element, and the two-dimensional protein nano array is formed by in-vitro self-assembly of an engineered S-layer on the surface of the semiconductor material. The biomolecule detection system comprises an HEMT sensor based on engineering S-layer in-vitro self-assembly, an electrical detection system and a signal processing and analyzing system.

Description

Technical Field

[0001] The present invention relates to the technical fields of nanobiology, biosensors, and biomolecule detection technologies, and in particular, to an engineered S-layer and a preparation method thereof, a HEMT sensor based on in vitro self-assembly of the engineered S-layer and a preparation method thereof, and a biomolecule detection system. Background Art

[0002] The outermost layer of the cell membranes of many bacteria and archaea contains a regular protein array, commonly known as the S-layer, which is one of the most abundant proteins in prokaryotic cells. S-layer protein monomers can self-assemble on various interfaces. After self-assembly, the S-layer can exhibit oblique (p1, p2), square (p4), or hexagonal (p3, p6) lattice symmetries, and the size of a single lattice is between 3-30 nm. Depending on the lattice type, each morphological unit consists of a different number of protein monomers.

[0003] The high-density two-dimensional lattice structure and excellent self-assembly properties of the S-layer have been widely applied in the fields of biomolecule immobilization, nanobiotechnology, and biosensing. Native S-layer proteins show great potential as pattern elements and nanoscale building blocks, and genetic engineering methods provide new ideas for changing the natural properties of S-layer proteins. After the insertion or fusion of functional protein domains in S-layer proteins, they can still maintain good self-assembly ability. Currently, a series of functional recombinant S-layer proteins have been designed through genetic engineering techniques, and these recombinant proteins have derived various novel affinity structures and biosensors, etc.

[0004] A field-effect transistor is an electronic device that controls current through the electric field effect, and among them, the high electron mobility field-effect transistor (HEMT) has been widely applied in the field of biosensing. The high electron mobility field-effect transistor is usually composed of semiconductor materials, and representative materials include gallium nitride, gallium arsenide, etc.

[0005] In 1969, scientists first synthesized gallium nitride (GaN) using hydride vapor phase epitaxy (HVPE) technology. Gallium nitride can be grown on substrates such as silicon (Si), silicon carbide (SiC), and sapphire, forming zinc blende and wurtzite crystals. The crystal structure of wurtzite gallium nitride is composed of hexagonal unit cells, and each hexagonal unit cell contains two adjacent hexagonal close-packed sublattices. In gallium nitride, the electronegativity of nitrogen is stronger than that of gallium. Nitrogen atoms will attract electrons from gallium atoms, resulting in an asymmetric charge distribution, making nitrogen partially negatively charged and gallium partially positively charged. The wurtzite structure of gallium nitride is non-centrosymmetric along the c-axis direction, and the centers of positive and negative charges do not coincide, resulting in polarization along the c-axis direction, which is called spontaneous polarization (Psp). The lattice constant of aluminum gallium nitride (AlGaN) is smaller than that of gallium nitride. When an AlGaN layer is grown on a GaN buffer layer, AlGaN will generate some tensile strain to adjust the lattice mismatch, and then this tensile strain leads to the piezoelectric polarization (Ppz) of the AlGaN layer. Opposite to spontaneous polarization, piezoelectric polarization is caused by external pressure. This strain causes the crystal to deform, thus generating a high strain-induced piezoelectric field. A heterostructure is formed between the two semiconductors AlGaN / GaN. Due to the difference in bandgap energy, the conduction band (EC) and valence band (EV) cannot be continuous at the interface. When the semiconductors are brought together, the Fermi level (Ef) will align and band bending will occur. The band bending will form a two-dimensional potential well on the surface of the heterojunction, and a large number of electrons will accumulate in this potential well. These electrons can only move two-dimensionally within the plane parallel to the heterojunction, so they are called two-dimensional electron gas (2DEG). The two-dimensional electron gas has extremely high electron mobility.

[0006] High electron mobility field effect transistor sensors show great application potential in ultraviolet detection, gas sensing, and biosensing.

[0007] Ultraviolet detectors have a wide range of applications in military and civilian fields, such as missile warning, fire warning, environmental ultraviolet detection, etc. The wide bandgap and robustness of gallium nitride are particularly suitable for ultraviolet detection. In recent years, new ultraviolet detectors based on gallium nitride, such as PIN diodes, Schottky diodes, and semiconductor-metal-based photodetectors based on gallium nitride, have been developed.

[0008] Gas sensors are mainly applied in fields such as automotive, aerospace, healthcare, and environmental protection. In gas sensors developed based on gallium nitride, the gas can catalyze the dissociation of the metal gate, thereby changing the charge distribution in the channel. Oxidizing gases such as NO2 and CO2 are easy to gain electrons, while reducing gases such as SO2, CO, NH3, and H2S are easy to lose electrons. The gain and loss of electrons will change the channel carrier concentration and the output drain current, and output as a sensing signal.

[0009] In addition, high electron mobility transistors (HEMTs) are also widely used in the field of biosensing. To detect the analyte to be analyzed, corresponding biosensing elements are modified on the gate (sensitive area). When the analyte binds to the biosensing element fixed on the sensing interface, the analyte will form an accumulation of positive or negative charges on the sensing interface, resulting in a change in the surface potential. At the same time, the accumulated charges will cause a change in the current in the channel, thus causing a change in the drain current and threshold voltage of the sensor. This change is output as a sensing signal.

[0010] HEMT-based biosensors have high sensitivity and high stability and have been applied to the detection of various small molecules, nucleic acids, pathogenic microorganisms, antibodies, and tumor markers. Currently, there have been studies that have achieved glucose detection with a minimum of 0.5 nM by immobilizing glucose oxidase on a HEMT sensor; TNF-α detection with a minimum of 10 -15 M by immobilizing aptamers; and spike protein detection with a minimum of 10 -22 M by immobilizing the spike protein antibody of the novel coronavirus.

[0011] However, the existing field-effect transistors still have the following technical defects in the application of biomolecule detection:

[0012] 1. Chemical cross-linking is often used to immobilize biosensing elements on HEMT sensors. The chemical cross-linking reaction first requires the activation of carboxyl groups and then the reaction with amino groups in biomolecules to achieve immobilization. Since these groups usually appear repeatedly in biomolecules, this immobilization method cannot achieve the directional immobilization of biosensing elements, thereby affecting the detection sensitivity and repeatability.

[0013] 2. Most chemical cross-linking reactions are not carried out under physiological conditions. Therefore, during the immobilization process, the loss of biomolecule activity is likely to occur, and the activity of the immobilized molecules is reduced, making it impossible to capture the analyte with high sensitivity.

[0014] 3. After immobilizing biosensing elements on HEMT sensors by chemical cross-linking methods, when detecting complex samples (plasma or serum), the non-specific binding signals on the sensor surface are relatively high.

[0015] Therefore, it is necessary to design a new engineered S-layer and apply it to a new biosensor and extend it to the entire biomolecule detection system to solve the problems existing in the prior art. Summary of the Invention

[0016] To solve the problems existing in the prior art, the present invention provides the following technical solutions: an engineered S-layer and its preparation method, a HEMT sensor based on the in vitro self-assembly of the engineered S-layer and its preparation method, and a biomolecule detection system. By utilizing the self-assembly ability of the recombinant S-layer protein, a two-dimensional protein nanoarray is constructed on a high electron mobility transistor by fusing the S-layer with a protein / polypeptide, thereby realizing the high-density and ordered immobilization of biomolecules. Through the ultrasensitive response of the S-layer functionalized HEMT sensor to surface charges, a high-performance sensing system is constructed to form a biomolecule detection system.

[0017] The first aspect of the present invention lies in providing an engineered S-layer formed by the fusion expression of a recombinant or redesigned S-layer protein and a biosensitive element, wherein the biosensitive element is one or more of an antibody protein, an antigen protein, or a polypeptide.

[0018] Preferably, the recombinant or redesigned S-layer protein includes one or more of the S-layer protein SbpA of Bacillus sphaericus, the S-layer protein SbsB of Geobacillus stearothermophilus, the S-layer protein SbsC of Bacillus stearothermophilus, the S-layer protein SgsE of Geobacillus stearothermophilus, and the S-layer protein HPI of Deinococcus radiodurans.

[0019] Preferably, the biosensitive element includes one or more of the nanobodies of angiotensin-converting enzyme 2 and the ovarian cancer antigen marker HE4, the streptavidin-binding peptide (SBP), the Avi-tag, and the Strep-TagII.

[0020] The second aspect of the present invention lies in providing a preparation method of the engineered S-layer of the first aspect, including:

[0021] (1) Constructing an expression vector of the engineered S-layer;

[0022] (2) Completing the expression of the engineered S-layer in Escherichia coli; and

[0023] (3) Purifying the engineered S-layer.

[0024] Preferably, constructing the expression vector of the engineered S-layer includes:

[0025] Constructing a vector for expressing the recombinant or redesigned S-layer protein in Escherichia coli, wherein pET28a is used as the vector backbone, and the recombinant or redesigned S-layer protein is connected with ACE2 or 1G8 through a flexible linker (GS)4 to form the expression vector.

[0026] Preferably, completing the expression of the engineered S-layer in Escherichia coli includes:

[0027] Transform the expression vector of the engineered S-layer constructed into Escherichia coli BL21 competent cells to obtain an expression strain of recombinant S-layer protein;

[0028] Inoculate the expression strain into LB medium with corresponding resistance and culture overnight under specific temperature conditions;

[0029] Transfer the solution after overnight culture at a ratio of 1% and continue to culture under specific temperature conditions until the OD 600 reaches 0.6 - 0.8;

[0030] Add 0.5 mM IPTG for induction, adjust the temperature to a specific temperature, and complete the expression of the engineered S-layer in Escherichia coli after inducing for a specific duration.

[0031] Preferably, purifying the engineered S-layer includes:

[0032] Under the first temperature condition, centrifuge the bacterial liquid after the expression of the engineered S-layer in Escherichia coli is completed with the first quantification for the first duration, and collect the induced bacterial cells;

[0033] Weigh the wet weight of the bacterial cells, resuspend a quantified amount of bacterial cells in a fixed volume of CaCl2, and let it stand on ice for the second duration;

[0034] Collect the bacterial cells again, resuspend them in a fixed volume of 50 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, and 1% glycerol with a pH of 9.0, add a quantified amount of lysozyme, and then place it in a water bath until the temperature rises to the second temperature condition;

[0035] Let the bacterial liquid stand in a water bath at the third temperature condition for the third duration, then add a fixed volume of 50 mM Tris-HCl, 150 mM NaCl, and 1% Triton X-100 with a pH of 9.0, let it stand at the fourth temperature condition for the fourth duration, and then use a high-pressure homogenizer to break the bacterial liquid. Centrifuge the second quantified amount of the broken bacterial liquid at the fifth duration and the fifth temperature condition, and discard the supernatant;

[0036] Wash the precipitate once with an appropriate amount of 50 mM Tris-HCl, 150 mM NaCl, and 1% Triton X-100 with a pH of 9.0; then, wash it twice with an appropriate amount of 50 mM Tris-HCl and 150 mM NaCl with a pH of 9.0, and discard the supernatant after centrifugation;

[0037] Weigh the wet weight of the washed precipitate, add 5 volumes of 50 mM Tris-HCl, 150 mM NaCl, and 5 M guanidine hydrochloride with a pH of 9.0, and shake it on ice for the sixth duration to fully dissolve it;

[0038] Centrifuge the third quantitative bacterial solution at the sixth temperature condition for the seventh duration, take the supernatant, and add an appropriate volume of 50 mM Tris-HCl with a pH of 9.0 and 150 mM NaCl to adjust the concentration of guanidine hydrochloride to 2 M;

[0039] Filter the supernatant and perform gel filtration chromatography using Superdex 200 Increase, and perform SDS-PAGE detection on the collected protein samples. After passing the SDS-PAGE detection index, the purified engineered S-layer is obtained.

[0040] The third aspect of the present invention lies in providing a HEMT sensor based on the in vitro self-assembly of the engineered S-layer, so as to form an S-layer functionalized HEMT sensor through the in vitro self-assembly of the engineered S-layer. The HEMT sensor includes:

[0041] A field effect transistor body and a gate sensitive region;

[0042] Among them, the field effect transistor body includes a source electrode and a drain electrode disposed on both sides;

[0043] The gate sensitive region is disposed between the source electrode and the drain electrode, and includes a semiconductor material for forming a heterojunction structure and a two-dimensional protein nanoarray containing a biosensitive element, wherein the two-dimensional protein nanoarray is formed by the in vitro self-assembly of the engineered S-layer on the surface of the semiconductor material.

[0044] Preferably, the in vitro self-assembly of the engineered S-layer is completed in a thin film chamber formed by polydimethylsiloxane (PDMS) of organosilicon elastomer to achieve high-density and orderly fixation of biomolecules.

[0045] Preferably, the heterojunction structure is a gallium nitride / aluminum gallium nitride heterojunction structure or a gallium arsenide / aluminum gallium arsenide heterojunction structure.

[0046] The fourth aspect of the present invention lies in providing a preparation method for the HEMT sensor of the third aspect, including:

[0047] S1, preparing the engineered S-layer based on the preparation method of the second aspect;

[0048] S2, preparing the HEMT sensor based on the engineered S-layer, including:

[0049] Obtain a high electron mobility transistor without in vitro self-assembly of the engineered S-layer, and irradiate it in an ultraviolet cleaning machine for the eighth duration;

[0050] Then wash it with piranha solution for the ninth duration, wherein the piranha solution is a solution formed by mixing H2SO4 and H2O2 in a volume ratio of 7:3;

[0051] The high electron mobility transistor after being cleaned with the piranha solution is cleaned again with ddH2O and then dried with nitrogen gas.

[0052] Adjust the solution concentration of the purified engineered S-layer to 1 mg / mL to prepare a buffer for in vitro self-assembly of the engineered S-layer.

[0053] Based on the buffer, the purified engineered S-layer is subjected to overnight in vitro self-assembly on the high electron mobility transistor without in vitro self-assembly of the engineered S-layer under the seventh temperature condition, thereby forming the HEMT sensor.

[0054] Preferably, the buffer for in vitro self-assembly of the engineered S-layer is 50 mM Tris-HCl, 150 mM NaCl, and 10 mM CaCl2 with a pH of 9.0.

[0055] The fifth aspect of the present invention lies in providing a biomolecule detection system based on the HEMT sensor of the third aspect, including:

[0056] The HEMT sensor with in vitro self-assembly of the engineered S-layer of the third aspect of the present invention, which is used to output the presence state and concentration of biomolecules in the analyte;

[0057] An electrical detection system, including a digital source meter and an automated control device, which is used for long-term real-time monitoring of the potential signal and recording of the dynamic change process;

[0058] A signal processing and analysis system, which is used to receive the electrical signal output by the digital source meter and output the characterization data for the presence state and concentration of the biomolecules in the analyte based on the processing and analysis of the electrical signal.

[0059] Preferably, the digital source meter is used for:

[0060] A. By applying a high-precision bias voltage or current to the HEMT sensor, ensuring that the field-effect transistor corresponding to the HEMT sensor operates stably in the linear or saturation region;

[0061] B. Measuring the change in surface potential caused by the binding of biomolecules to the biosensitive elements in the two-dimensional protein nanoarray on the surface of the HEMT sensor, thereby converting the biological reaction after the binding of the biomolecules in the analyte to the biosensitive elements in the two-dimensional protein nanoarray on the surface of the HEMT sensor into a quantifiable electrical signal.

[0062] C. Scan the gate-source voltage and generate an output characteristic curve or a transfer characteristic curve based on the gate-source voltage; wherein, the influence of the binding of the biomolecules in the analyte to the biosensitive elements in the two-dimensional protein nanoarray on the HEMT sensor surface on the threshold voltage can be analyzed and determined through the output characteristic curve or the transfer characteristic curve; and the presence state and concentration of the biomolecules in the analyte can be determined through the shift of the output characteristic curve or the transfer characteristic curve.

[0063] Preferably, the change in the surface potential is characterized by a change in at least one of the source-drain current, the source-drain voltage, and the gate-source voltage.

[0064] Preferably, the field effect transistor is a junction field effect transistor (junction FET-JFET).

[0065] Preferably, the processing and analysis include:

[0066] Analyze and determine the influence of the binding of the biomolecules in the analyte to the biosensitive elements in the two-dimensional protein nanoarray on the HEMT sensor surface on the threshold voltage through the output characteristic curve or the transfer characteristic curve; and

[0067] Determine the presence state and concentration of the biomolecules in the analyte through the shift of the output characteristic curve or the transfer characteristic curve.

[0068] The S-layer protein, the HEMT sensor, and the biomolecule detection system provided by the present invention at least have the following beneficial effects:

[0069] (1) The fixation amount of the biosensitive elements on the HEMT sensor surface is increased. This is because the biomolecules are fixed on the sensing interface through the self-assembly of the recombinant S-layer protein, forming a two-dimensional nanoarray of proteins, thus increasing the fixation amount.

[0070] (2) Compared with chemical cross-linking, the non-specific binding signal on the sensor surface is reduced. This is because the dense protein film formed by the self-assembly of the S-layer can effectively reduce the non-specific binding signal on the sensor surface and shows good anti-fouling properties for both single proteins and plasma samples.

[0071] (3) Detection of HE4 protein at 10 -22 M is achieved, and the linear detection range is from 10 -22 M to 10 -14 M, and it can effectively distinguish tumor patients from healthy people in the detection of clinical samples. Description of the Drawings

[0072] Figure 1This is the schematic diagram of the principle architecture of a HEMT (High Electron Mobility Transistors) sensor based on engineered S-layer in vitro self-assembly according to the present invention.

[0073] Figure 2 This is the schematic diagram of the principle architecture of a biomolecule detection system based on a HEMT sensor provided by the present invention.

[0074] Figure 3 In (a) is a schematic diagram of the amount of protein immobilized by self-assembly of S-layer protein for rSbpA-1G8 with the same concentration. Figure 3 In (b) is a schematic diagram of the amount of protein immobilized by chemical cross-linking for rSbpA-1G8 with the same concentration.

[0075] Figure 4 In (a) is a schematic diagram for evaluating the non-specific binding signal on the gold surface after immobilizing protein by engineered S-layer self-assembly using surface plasmon resonance. Figure 4 In (b) is a schematic diagram for evaluating the non-specific binding signal on the gold surface after immobilizing protein by chemical cross-linking using surface plasmon resonance.

[0076] Figure 5 This is a schematic diagram of the transfer characteristic curves at various concentrations in the case of detecting HE4 protein by an S-layer-based HEMT immunosensor according to the present invention.

[0077] Figure 6 This is the regression curve of HE4 concentration / BSA concentration versus the corresponding potential offset value according to the present invention.

[0078] Figure 7 This is a schematic diagram of detecting clinical samples by an S-layer-based HEMT sensor according to the present invention. Detailed implementation manners

[0079] To better understand the above technical solutions, the above technical solutions will be described in detail below in conjunction with the accompanying drawings of the specification and specific implementation manners.

[0080] The method provided by the present invention can be implemented in the following terminal environment. The terminal may include one or more of the following components: a processor, a memory, and a display screen. Among them, at least one instruction is stored in the memory, and the instruction is loaded and executed by the processor to implement the method described in the following embodiments.

[0081] The processor may include one or more processing cores. The processor connects various parts inside the entire terminal through various interfaces and lines, and by running or executing instructions, programs, code sets, or instruction sets stored in the memory, and by calling data stored in the memory, it executes various functions of the terminal and processes data.

[0082] The memory may include a Random Access Memory (RAM), or may also include a Read-Only Memory (ROM). The memory can be used to store instructions, programs, codes, code sets or instructions.

[0083] The display screen is used to display the user interfaces of various application programs.

[0084] In addition, those skilled in the art can understand that the structure of the above terminal does not constitute a limitation on the terminal. The terminal may include more or fewer components, or combine certain components, or have different component arrangements. For example, the terminal also includes components such as a radio frequency circuit, an input unit, a sensor, an audio circuit, and a power supply, which will not be elaborated here.

[0085] Embodiment 1

[0086] This embodiment provides an engineered S-layer, which is formed by the fusion expression of a recombinant or redesigned S-layer protein and a biosensing element, wherein the biosensing element is one or more of an antibody protein, an antigen protein or a polypeptide.

[0087] As a preferred embodiment, the recombinant or redesigned S-layer protein includes: one or more of SbpA, the S-layer protein of Bacillus sphaericus; SbsB, the S-layer protein of Geobacillus stearothermophilus; SbsC, the S-layer protein of Bacillus stearothermophilus; SgsE, the S-layer protein of Geobacillus stearothermophilus; and HPI, the S-layer protein of Deinococcus radiodurans.

[0088] As a preferred embodiment, the biosensing element includes one or more of a nanobody against angiotensin-converting enzyme 2 (ACE2) and ovarian cancer antigen marker HE4, a streptavidin-binding peptide (SBP), an Avi-tag, and a Strep-TagII.

[0089] In this embodiment, the redesign of the S-layer protein is achieved by fusing and expressing it with other proteins (antigens, antibodies) to construct a two-dimensional nanoarray of antigens / antibodies. Through recombination or redesign, the S-layer protein is fused and expressed with the biosensing element to form an engineered S-layer. The engineered S-layer can self-assemble into a two-dimensional nanoarray on the gold surface / sensing interface, thereby realizing the high-density and ordered immobilization of the biosensing element and the highly sensitive detection of the analyte.

[0090] Embodiment 2

[0091] This embodiment provides a preparation method for the engineered S-layer of the first aspect, including:

[0092] (1) Construct the expression vector of the engineered S-layer;

[0093] In this example, the construction of the expression vector of the engineered S-layer includes:

[0094] Construct a vector in Escherichia coli to express the recombinant or redesigned S-layer protein. Among them, pET28a is used as the vector backbone, and the recombinant or redesigned S-layer protein is connected with ACE2 or 1G8 through a flexible linker (GS)4 to form the expression vector.

[0095] (2) Complete the expression of the engineered S-layer in Escherichia coli;

[0096] As a preferred embodiment, the completion of the expression of the engineered S-layer in Escherichia coli includes:

[0097] Transform the constructed expression vector of the engineered S-layer into Escherichia coli BL21 competent cells to obtain an expression strain of the recombinant S-layer protein;

[0098] Inoculate the expression strain into an LB medium with corresponding resistance and culture it overnight at 37°C;

[0099] Transfer the solution after overnight culture at a ratio of 1% and continue to culture at 37°C until the OD 600 reaches 0.6 - 0.8;

[0100] Add 0.5 mM IPTG for induction, adjust the temperature to 16°C, and complete the expression of the engineered S-layer in Escherichia coli after 18 hours of induction.

[0101] (3) Purify the engineered S-layer;

[0102] As a preferred embodiment, the purification of the engineered S-layer includes:

[0103] Under the condition of 4°C, centrifuge the bacterial liquid after the expression of the engineered S-layer in Escherichia coli at 4000g for 10 min to collect the induced bacterial cells;

[0104] Weigh the wet weight of the bacterial cells, resuspend 1 g of the bacterial cells in 100 mL of 0.1 M CaCl2, and let it stand on ice for 30 min;

[0105] Collect the bacterial cells again, resuspend them in 100 mL of 50 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA and 1% glycerol with a pH of 9.0, add 10 mg of lysozyme, and then place it in a water bath to incubate until the temperature rises to 42°C;

[0106] The bacterial solution was left standing in a 30 °C water bath for 15 min, then 10 mL of 50 mM Tris-HCl with a pH of 9.0, 150 mM NaCl, and 1% Triton X-100 were added, and it was left standing in a 20 °C environment for 10 min. Subsequently, the bacterial solution was disrupted using a high-pressure homogenizer, and the disrupted bacterial solution was centrifuged at 20,000 g for 30 min at 4 °C, and the supernatant was discarded.

[0107] The precipitate was washed once with an appropriate amount of 50 mM Tris-HCl with a pH of 9.0, 150 mM NaCl, and 1% Triton X-100; it was washed twice with an appropriate amount of 50 mM Tris-HCl with a pH of 9.0 and 150 mM NaCl, and the supernatant was discarded after centrifugation.

[0108] The wet weight of the washed precipitate was weighed, and 5 volumes of 50 mM Tris-HCl with a pH of 9.0, 150 mM NaCl, and 5 M guanidine hydrochloride were added, and it was shaken on ice for 20 min to fully dissolve it.

[0109] The bacterial solution at 36,000 g was centrifuged at 4 °C for 30 min, and the supernatant was taken. An appropriate volume of 50 mM Tris-HCl with a pH of 9.0 and 150 mM NaCl was added to adjust the concentration of guanidine hydrochloride to 2 M.

[0110] The supernatant was filtered and then subjected to gel filtration chromatography using Superdex 200 Increase, and the collected protein samples were detected by SDS-PAGE. After passing the SDS-PAGE detection index, the purified engineered S-layer was obtained.

[0111] Example 3

[0112] As Figure 1 shown, this example provides a HEMT (High Electron Mobility Transistors) sensor based on the in vitro self-assembly of the engineered S-layer in Example 1. Thus, an S-layer functionalized HEMT sensor is formed through the in vitro self-assembly of the engineered S-layer, which is used for the ultrasensitive response to surface charges to construct a high-performance sensing system. The HEMT sensor includes:

[0113] A field-effect transistor body and a gate sensitive region;

[0114] Among them, the field-effect transistor body includes a source and a drain disposed on both sides;

[0115] The gate sensitive region is disposed between the source and the drain, and includes a semiconductor material for forming a heterojunction structure and a two-dimensional protein nanoarray containing a biosensing element, wherein the two-dimensional protein nanoarray is formed by in vitro self-assembly of the engineered S-layer on the surface of the semiconductor material.

[0116] PDMA is used to form a closed liquid environment, and a silver / silver chloride reference electrode is added to form a liquid gate. The surface of the HEMT sensor is modified with a gold film, and the engineered S-layer can be self-assembled in vitro on the gold film.

[0117] The semiconductor material in the sensitive region forms a heterojunction structure, and the two-dimensional electron gas on the surface of the heterojunction structure is very sensitive to charge changes. When the analyte binds to the biosensing element on the surface of the HEMT sensor, it will cause a change in the surface potential. The change in potential is output as a potential signal, and the electrical detection system collects and outputs the potential signal, and the presence state and concentration of the analyte are judged by analyzing the change in potential.

[0118] In this embodiment, the heterojunction structure is a gallium nitride / aluminum gallium nitride heterojunction structure or a gallium arsenide / aluminum arsenide gallium heterojunction structure, so as to form a high electron mobility field effect transistor. However, in practical applications, it is not limited to this specific high electron mobility field effect transistor sensor. Those skilled in the art can also select other types of high electron mobility field effect transistors containing heterojunction structures, as long as they are suitable for in vitro self-assembly of the engineered S-layer, they are within the protection scope of the present invention.

[0119] The working principle of the HEMT sensor (taking the heterojunction structure as the gallium nitride / aluminum gallium nitride (GaN / AlGaN) heterojunction structure as an example):

[0120] On the surface of the HEMT sensor, when the analyte binds to the biosensing element, it will change the potential distribution on the surface of the GaN / AlGaN heterojunction structure, thereby causing a change in the channel resistance. This change can be characterized by the shift of the output characteristic curve or the transfer characteristic curve and can be used for the detection of the presence state and concentration of biomolecules in the analyte.

[0121] Example 4

[0122] This embodiment provides a preparation method of the HEMT sensor in the third aspect, including:

[0123] S1, preparing the engineered S-layer based on the preparation method of Example 2;

[0124] S2, preparing the HEMT sensor based on the engineered S-layer, including:

[0125] Obtain a high electron mobility transistor with non-engineered S-layer self-assembled in vitro, and place it in an ultraviolet cleaning machine for irradiation for 15 min;

[0126] Then clean it with piranha solution for 1 min, where the piranha solution is a solution formed by mixing H2SO4 and H2O2 in a volume ratio of 7:3;

[0127] Use a large amount of ddH2O to clean the high electron mobility transistor again after being cleaned with the piranha solution, and then dry it with nitrogen;

[0128] Adjust the solution concentration of the purified engineered S-layer to 1 mg / mL, and configure a buffer for in vitro self-assembly of the engineered S-layer;

[0129] Based on the buffer, perform overnight in vitro self-assembly of the purified engineered S-layer on the high electron mobility transistor with non-engineered S-layer self-assembled in vitro at 4 °C, so as to form the HEMT sensor.

[0130] In this embodiment, the buffer for in vitro self-assembly of the engineered S-layer is a mixed solution composed of a weak acid and its salt, and a weak base and its salt, which can offset and reduce the influence of externally added strong acid or strong base on the solution acidity to a certain extent, so as to keep the pH value of the solution relatively stable.

[0131] As a preferred implementation manner, the buffer for in vitro self-assembly of the engineered S-layer is 50 mM Tris-HCl, 150 mM N3Cl and 10 mM CaCl2 with a pH of 9.0.

[0132] In this embodiment, the HEMT sensor based on in vitro self-assembly of the engineered S-layer can be widely used in biomolecule detection. This is because after the recombinant S-layer protein completes self-assembly on the HEMT sensor, a two-dimensional nanoarray of biosensing elements is formed. The high-density and orderly fixed biosensing elements can be highly sensitively captured by the analytes in the sample.

[0133] Example Five

[0134] As shown in Figure 2 This embodiment provides a biomolecule detection system based on the HEMT sensor of Example Three, including:

[0135] The engineered S-layer self-assembled HEMT sensor according to the third aspect of the present invention is used to output the presence state and concentration of biomolecules in the analyte; in this embodiment, when the biomolecules in the analyte bind to the biosensing elements in the two-dimensional protein nanoarray on the surface of the HEMT sensor, it causes a change in the surface potential, and the change in potential is output as a potential signal, and the potential signal is used to reflect the presence state and concentration of the biomolecules in the analyte;

[0136] An electrical detection system, including a digital source meter and an automated control device, is used for long-term real-time monitoring and recording of the dynamic change process of the potential signal, and its working principle includes: the digital source meter is used for:

[0137] A. By applying a high-precision bias voltage or current to the HEMT sensor, ensuring that the field effect transistor (abbreviated as FET, hereinafter referred to as field effect transistor) corresponding to the HEMT sensor operates stably in the linear or saturation region;

[0138] B. Measuring the change in the surface potential caused by the binding of biomolecules to the biosensing elements in the two-dimensional protein nanoarray on the surface of the HEMT sensor, and the change in the surface potential is characterized by a change in at least one of the source-drain current, source-drain voltage, and gate-source voltage, so as to convert the biological reaction after the biomolecules in the analyte bind to the biosensing elements in the two-dimensional protein nanoarray on the surface of the HEMT sensor into a quantifiable electrical signal;

[0139] C. Scanning the gate-source voltage and generating an output characteristic curve or a transfer characteristic curve based on the gate-source voltage; among them, through the output characteristic curve or the transfer characteristic curve, the influence of the binding of the biomolecules in the analyte to the biosensing elements in the two-dimensional protein nanoarray on the surface of the HEMT sensor on the threshold voltage can be analyzed and determined; and the presence state and concentration of the biomolecules in the analyte can be determined through the offset of the output characteristic curve or the transfer characteristic curve.

[0140] As a preferred embodiment, the field effect transistor is a junction field effect transistor (JFET).

[0141] A signal processing and analysis system is used to receive the electrical signal output by the digital source meter, and based on the electrical signal, perform signal processing and analysis through relevant software, and then output the characterization data for the presence state and concentration of the biomolecules in the analyte.

[0142] Experimental examples and effect verification:

[0143] (1) The immobilization amount of the bioreceptor on the surface of the HEMT sensor is increased. This is because biomolecules are immobilized on the sensing interface through the self-assembly of recombinant S-layer protein, forming a two-dimensional nanoarray of proteins, thus increasing the immobilization amount.

[0144] Taking the fusion protein rSbpA-1G8 of S-layer protein SbpA from Bacillus sphaericus and nanobody 1G8 as an example, at the same concentration of rSbpA-1G8, it is immobilized by the self-assembly of S-layer protein and chemical cross-linking method respectively. The immobilization amounts are as Figure 3 shown in (a) and (b) of []. The self-assembly of S-layer protein can achieve the protein immobilization of 5052RU, while chemical cross-linking can only immobilize 3414.8RU. That is, the protein amount immobilized by the self-assembly of S-layer protein is 50% higher than that of chemical cross-linking.

[0145] (2) Compared with chemical cross-linking, the non-specific binding signal on the sensor surface is reduced. This is because the dense protein film formed by S-layer self-assembly can effectively reduce the non-specific binding signal on the sensor surface and shows good anti-fouling properties for both single protein and plasma samples.

[0146] In this embodiment, surface plasmon resonance is used to evaluate the non-specific binding signal on the gold surface. Plasma and serum with a dilution of 1:10 are respectively passed over the gold surface after protein immobilization. The results are as Figure 4 shown in (a) and (b) of []. The sensing surface formed by the self-assembly of S-layer protein shows excellent anti-fouling properties. Specifically, when detecting plasma on the sensing surface formed by the self-assembly of S-layer protein, the RU value increases by 34.6, and when detecting serum, the RU value increases by 41.4. While under the same detection conditions after immobilizing proteins by chemical cross-linking method, the RU values increase by 90.3 and 74.9 respectively. This shows that compared with the chemical cross-linking method, the S-layer-based immobilization method reduces the non-specific binding signal by 62% in plasma detection and 45% in serum detection. This result indicates that the sensing interface obtained by the self-assembly of S-layer protein can more effectively reduce the non-specific binding signal on the chip surface because a dense protein film is formed after the self-assembly of rSbpA-1G8 on the chip surface, thus maintaining good anti-fouling properties in the detection of complex samples.

[0147] (3) The detection of HE4 protein at 10 -22 M is achieved, and the linear detection range is from 10 -22 M to 10 -14 M, which can effectively distinguish tumor patients from healthy people in the detection of clinical samples.

[0148] Detection of ovarian cancer antigen HE4 using an S-layer-based HEMT sensor. Different concentrations of HE4 protein were sequentially added to the HEMT sensor, and the transfer characteristic curves at each concentration were recorded.

[0149] The cut-off value for HE4 protein detection was defined as three times the maximum potential shift value generated by the negative protein (BSA), i.e., 25.98 mV ( Figure 5 the uppermost dotted line in -22 . The potential shift value generated by 10 -22 M of HE4 was 28.33 mV. Therefore, the detection limit of HE4 protein was found to be 10 Figure 6 M. The regression curve of HE4 concentration versus the corresponding potential shift value was plotted as shown in -22 and linear regression analysis was performed. The results showed that in the concentration range of 10 -14 M to 10 2 M, the potential shift value was linearly correlated with the HE4 concentration, with R

[0150] Using an S-layer-based HEMT immunosensor, plasma samples from normal individuals and ovarian cancer patients were tested, including 30 ovarian cancer patients and 20 healthy individuals. The test results are shown in Figure 7 . The potential shifts generated by ovarian cancer patient samples were generally higher than those of normal individuals. The average potential shift value generated by 20 healthy individual samples was 21.07 mV, while the average potential shift value generated by 30 ovarian cancer plasma samples was 136.2 mV, with a maximum of 237.7 mV.

[0151] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention. Obviously, those skilled in the art can make various changes and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. An engineered S-layer, characterized in that, The engineered S-layer is formed by the fusion expression of a recombinant or redesigned S-layer protein with a biosensing element, wherein the biosensing element is one or more of an antibody protein, an antigen protein, or a polypeptide.

2. An engineered S-layer according to claim 1, characterized in that, The recombinant or redesigned S-layer protein includes one or more of the S-layer protein SbpA of Bacillus sphaericus, the S-layer protein SbsB of Geobacillus stearothermophilus, the S-layer protein SbsC of Bacillus stearothermophilus, the S-layer protein SgsE of Geobacillus stearothermophilus, and the S-layer protein HPI of Deinococcus radiodurans.

3. An engineered S-layer according to claim 2, characterized in that, The biosensing element includes one or more of angiotensin-converting enzyme 2, a nanobody against the ovarian cancer antigen biomarker HE4, streptavidin-binding peptide (SBP), Avi-tag, and Strep-TagII.

4. The method for preparing the engineered S-layer according to any one of claims 1-3, characterized in that, Including: (1) Construct an expression vector for the engineered S-layer; (2) Achieve the expression of the engineered S-layer in Escherichia coli; And (3) Purify the engineered S-layer.

5. The preparation method of the engineered S-layer according to claim 4, characterized in that, The construction of the expression vector for the engineered S-layer includes: Construct a vector for expressing the recombinant or redesigned S-layer protein in Escherichia coli. Among them, pET28a is used as the vector backbone, and the recombinant or redesigned S-layer protein is linked to ACE2 or the nanobody through a flexible linker (GS)4 to form the expression vector.

6. The preparation method of the engineered S-layer according to claim 5, characterized in that Achieving the expression of the engineered S-layer in Escherichia coli includes: Transform the constructed expression vector of the engineered S-layer into Escherichia coli BL21 competent cells to obtain an expression strain of the recombinant S-layer protein; Inoculate the expression strain into an LB medium with the corresponding resistance and culture it overnight at a specific temperature condition; Transfer the solution after overnight culture at a ratio of 1% and continue to culture it under specific temperature conditions until OD 600 reaches 0.6 - 0.8; Add 0.5 mM IPTG for induction, adjust the temperature to a specific temperature, and complete the expression of the engineered S-layer in Escherichia coli after inducing for a specific duration.

7. The preparation method of the engineered S-layer according to claim 6, characterized in that, Purifying the engineered S-layer includes: At the first temperature condition, centrifuge the bacterial liquid after the expression of the engineered S-layer in Escherichia coli is completed with the first quantification for the first duration, and collect the induced bacterial cells; Weigh the wet weight of the bacterial cells, resuspend a quantified amount of the bacterial cells in a fixed volume of CaCl2, and let it stand on ice for the second duration; Collect the bacterial cells again, resuspend them in a fixed volume of 50 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, and 1% glycerol with a pH of 9.0, add a quantified amount of lysozyme, and then place it in a water bath until the temperature rises to the second temperature condition; Let the bacterial liquid stand in a water bath at the third temperature condition for the third duration, then add a fixed volume of 50 mM Tris-HCl, 150 mM NaCl, and 1% Triton X-100 with a pH of 9.0, let it stand at the fourth temperature condition for the fourth duration, and then use a high-pressure homogenizer to break the bacterial liquid. Centrifuge the second quantified amount of the broken bacterial liquid at the fifth duration and the fifth temperature condition, and discard the supernatant. Wash the precipitate once with an appropriate amount of 50 mM Tris-HCl, 150 mM NaCl, and 1% Triton X-100 at pH 9.0; then, wash it twice with an appropriate amount of 50 mM Tris-HCl and 150 mM NaCl at pH 9.0, discard the supernatant after centrifugation; Weigh the wet weight of the washed precipitate, add 5 volumes of 50 mM Tris-HCl, 150 mM NaCl, and 5 M guanidine hydrochloride at pH 9.0, and shake it on ice for six hours to fully dissolve it; Centrifuge the third quantitative bacterial solution for seven hours under the sixth temperature condition, take the supernatant, and adjust the concentration of guanidine hydrochloride to 2 M by adding an appropriate volume of 50 mM Tris-HCl and 150 mM NaCl at pH 9.0; Filter the supernatant and perform gel filtration chromatography using Superdex 200 Increase, and perform SDS-PAGE detection on the collected protein samples. After passing the SDS-PAGE detection index, the purified engineered S-layer is obtained.

8. The engineered S-layer in vitro self-assembled HEMT sensor according to any one of claims 1-3, whereby an S-layer functionalized HEMT sensor is formed by the engineered S-layer in vitro self-assembly, characterized in that, The HEMT sensor includes: A field effect transistor body and a gate sensitive region; Among them, the field effect transistor body includes a source electrode and a drain electrode disposed on both sides; The gate sensitive region is disposed between the source electrode and the drain electrode, and includes a semiconductor material for forming a heterojunction structure and a two-dimensional protein nanoarray containing a bio-sensitive element, wherein the two-dimensional protein nanoarray is formed by in vitro self-assembly of the engineered S-layer on the surface of the semiconductor material.

9. The engineered S-layer self-assembled in vitro HEMT sensor according to claim 8, characterized in that Complete the in vitro self-assembly of the engineered S-layer in a thin film chamber formed by organosilicon elastomer polydimethylsiloxane to achieve high-density and ordered fixation of biomolecules.

10. The engineered S-layer self-assembled in vitro HEMT sensor according to claim 9, wherein The heterojunction structure is a gallium nitride / aluminum gallium nitride heterojunction structure or a gallium arsenide / aluminum arsenide gallium heterojunction structure.

11. The preparation method of the HEMT sensor according to any one of claims 8-10, characterized in that, Include: S1, Prepare an engineered S-layer, and the engineered S-layer is prepared by the method described in any one of claims 4-7; S2, Prepare the HEMT sensor based on the engineered S-layer, including: Obtain a high electron mobility transistor without in vitro self-assembly of the engineered S-layer, and irradiate it in an ultraviolet cleaning machine for eight hours; Then wash it with piranha solution for nine hours, wherein the piranha solution is a solution formed by mixing H2SO4 and H2O2 in a volume ratio of 7:3; Wash the high electron mobility transistor after being washed with the piranha solution again with ddH2O and then blow it dry with nitrogen; Adjust the solution concentration of the purified engineered S-layer to 1 mg / mL, and configure a buffer solution for in vitro self-assembly of the engineered S-layer; Based on the buffer solution, perform overnight in vitro self-assembly of the purified engineered S-layer on the high electron mobility transistor without in vitro self-assembly of the engineered S-layer under the seventh temperature condition to form the HEMT sensor.

12. The preparation method of the HEMT sensor according to claim 11, characterized in that, The buffer solution for in vitro self-assembly of the engineered S-layer is 50 mM Tris-HCl, 150 mM NaCl, and 10 mM CaCl2 at pH 9.

0.

13. A biomolecule detection system, characterized in that, Comprising: The HEMT sensor based on in vitro self-assembly of engineered S-layer according to any one of claims 8-10, which is used to output the presence state and concentration of biomolecules in the analyte; An electrical detection system, including a digital source meter and an automatic control device, which is used for long-time real-time monitoring of the potential signal and recording of the dynamic change process; A signal processing and analysis system, which is used to receive the electrical signal output by the digital source meter and output the characterization data of the presence state and concentration of the biomolecules in the analyte based on the processing and analysis of the electrical signal.

14. A biomolecule detection system according to claim 13, characterized in that, The digital source meter is used for: A. Ensuring that the field effect transistor corresponding to the HEMT sensor operates stably in the linear or saturation region by applying a high-precision bias voltage or current to the HEMT sensor; B. Measuring the change in surface potential caused by the binding of biomolecules to the biosensitive elements in the two-dimensional protein nanoarray on the surface of the HEMT sensor, so as to convert the biological reaction after the binding of the biomolecules in the analyte to the biosensitive elements in the two-dimensional protein nanoarray on the surface of the HEMT sensor into a quantifiable electrical signal; C. Scanning the gate-source voltage and generating an output characteristic curve or a transfer characteristic curve based on the gate-source voltage; wherein, the influence of the binding of the biomolecules in the analyte to the biosensitive elements in the two-dimensional protein nanoarray on the surface of the HEMT sensor on the threshold voltage can be analyzed and determined through the output characteristic curve or the transfer characteristic curve; and the presence state and concentration of the biomolecules in the analyte can be determined through the offset of the output characteristic curve or the transfer characteristic curve.

15. A biomolecule detection system according to claim 14, characterized in that, The change in the surface potential is characterized by a change in at least one of the source-drain current, the source-drain voltage, and the gate-source voltage.

16. A biomolecule detection system according to claim 15, wherein, The field effect transistor is a junction field effect transistor.

17. A biomolecule detection system according to claim 16, characterized in that, The processing and analysis include: Analyzing and determining the influence of the binding of the biomolecules in the analyte to the biosensitive elements in the two-dimensional protein nanoarray on the surface of the HEMT sensor on the threshold voltage through the output characteristic curve or the transfer characteristic curve; and Determining the presence state and concentration of the biomolecules in the analyte through the offset of the output characteristic curve or the transfer characteristic curve.

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